2022
UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO BIOLOGIA VEGETAL
Reappraising the use of beta-lactam antibiotics against tuberculosis: study of genes associated with beta-lactam
susceptibility in mycobacteria
Diana da Costa Mortinho
Mestrado em Microbiologia Aplicada
Dissertação orientada por:
Professora Doutora Maria João Catalão
Professora Doutora Mónica V. Cunha
2022
Professor Mónica V. Cunha was the internal supervisor designated in the scope of the Master in Applied Microbiology of the Faculty of Sciences of the University of Lisbon.
This Dissertation was fully performed at Faculdade de Farmácia da Universidade de Lisboa under the direct supervision of Professor Maria João Catalão and co-supervision of Francisco
Olivença and Cátia Silveiro.
I
Acknowledgments
First and foremost, I would like to thank Drª. Maria João Catalão who trusted me with this project, providing me with invaluable advice and motivation that contributed tremendously to the successful completion of the project.
Secondly, I want to thank Cátia and Francisco for everything. I’m very grateful and honoured for all the teachings you gave me. These were stressful and challenging times but with your support, all went well. I’m perfectly aware that you made me a better scientist and I’m one hundred percent sure that you both are the best scientists I will ever know. I hope everything goes well with your PhD and that you both find and live true happiness with your work and life.
Besides, I would like to thank all members of the Pathogen Genome Bioinformatics and Computational Biology research group whom I shared equipment, reagents, and room space. Thank you for making me feel welcomed and for all the help. Specially, I would like to thank Luís for the all the advice and jokes, Tiago and Ricardo for always putting my inoculums inside the incubator, and Leonor for all the laughs, cries, help and support.
Additionally, I would like to thank Joana and Sofia for letting me occupy your laboratory and use your equipment, several times, and for all the support and laughs you gave me through the time of this project. I hope you do well in your next steps and find enjoyment after your projects.
At last, but not least, I would like to thank my family and friends for their support. Without it I couldn’t have succeeded in completing this project. Specially, I thank my mom and my sister for giving me all the support that I could have asked. Also, my biggest fan and supporter, my Daniel whom I really can’t thank enough for all the sincere love. Additionally, I would like to thank all my friends in weeb kimchi with whom I share a path in academic studies. Thank you for the support, laughs, games, and true friendship.
This project wouldn’t be possible without all of you. I invested all my physical and mental energy into this project, and I hope to make everyone here proud as much as I’m of this project.
This work was supported by funds provided by Fundação para a Ciência e Tecnologia (PTDC/BIA-MIC/31233/2017 and IF/00414/2015 to Maria João Catalão) and by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2018 – to Maria João Catalão).
II
Abstract
Mycobacterium tuberculosis (Mtb) causes one of the deadliest diseases in the world, tuberculosis (TB). The emergence of Mtb strains that are both multi- and extensively drug-resistant (MDR/XDR) emphasizes the urgent need to create novel drugs and efficient treatment modalities.
β-lactams are a bactericidal class of antibiotics frequently used in medical practice and the chosen treatment for a wide range of infections, caused by various pathogens. The rise of MDR-TB has encouraged renewed interest in β-lactam/β-lactamase inhibitor combination therapy. The application of β-lactams still has limitations, with further studies being necessary to prove their clinical benefit. The identification of genomic variants associated with differential susceptibility to β-lactams is particularly relevant for this. A recent study revealed a group of putative genomic markers of differential β-lactam phenotypes, which includes the lpqK (Rv0339c) gene. Moreover, this study uncovered that mutations in peptidoglycan (PG) hydrolase genes like cwlM (Rv3915) that were associated with lower β-lactam minimum inhibitory concentrations (MICs).
To understand the role of cwlM and lpqK for a phenotype of susceptibility to β-lactams, knockdown mutants were constructed in the mycobacterial study model, Mycolicibacterium smegmatis (M. smegmatis), using the clustered regularly interspaced short palindromic repeat interference (CRISPRi) system. It’s a straightforward and cost-effective platform to repress the expression of genes.
Characterization assays of both knockdown mutants confirmed the essentiality of cwlM and the non- essentiality of lpqK and the repression of cwlM impacts the susceptibility of M. smegmatis to cefotaxime and meropenem, while lpqK repression does not. Protein characterization studies revealed that CwlMMtb
does not have PG-hydrolytic activity and that LpqKMtb has the highest nitrocefin hydrolysis activity.
Synergy assays with β-lactams and CwlMMtb revealed that it hinders the bactericidal activity of cefotaxime against M. smegmatis. These findings contribute significantly to future understanding of mycobacterial pathogenesis and the development of alternative TB therapeutics with β-lactams.
Keywords: Tuberculosis; Antibiotic resistance; CRISPRi; PG hydrolase; β-lactams
III
Resumo
Mycobacterium tuberculosis (Mtb) é o patógeno intracelular que causa uma das doenças mais mortais do mundo, a tuberculose (TB), chegando aos 1,5 milhões de óbitos associados por ano.
Normalmente, a TB ativa e Mtb suscetível aos antibióticos tem um tratamento padrão de seis meses com a administração concomitante de quatro agentes antituberculose (isoniazida, pirazinamida, etambutol, rifampicina). As resistências surgem quando os medicamentos anti-TB são usados de forma inadequada, por meio de prescrição incorreta por parte dos profissionais de saúde, baixa qualidade dos antimicrobianos, interrupção das cadeias de transporte e baixa adesão do doente. O aumento de estirpes de Mtb que são multi- e extensivamente resistentes a antimicrobianos (MDR e XDR) enfatiza a necessidade urgente de novos agentes anti-TB e esquemas terapêuticos mais eficientes.
O envelope de Mtb é uma estrutura complexa. A estrutura essencial da parede celular é composta por três componentes principais: um polímero reticulado de peptidoglicano (PG), um polissacarídeo altamente ramificado de arabinogalactano (AG) e ácidos micólicos de cadeia longa (MA). Estes componentes formam o complexo micol-arabinogalactano-peptidoglicano (mAGP). Sendo a principal característica do envelope celular micobacteriano, o complexo mAGP é responsável pela resistência inata a muitos antibióticos comumente usados e também está envolvido na virulência de Mtb. As enzimas que catalisam a biossíntese e reciclagem de componentes da parede celular, em particular do PG, são essenciais para a viabilidade de Mtb, e, portanto, são alvos atraentes para o desenvolvimento de novos antibióticos.
Os β-lactâmicos são uma classe de antibióticos bactericidas, sendo os antibióticos mais bem validados na prática médica e o tratamento de escolha para uma ampla gama de infeções, causadas por vários patógenos bacterianos. Essencialmente, os β-lactâmicos têm como mecanismo de ação a inibição das transpeptidases do PG. A sua exclusão do tratamento da TB é notável, mas o aumento da MDR-TB estimulou um interesse renovado na terapia combinada de inibidores de β-lactamases com β-lactâmicos.
A aplicação de β-lactâmicos no tratamento da TB ainda apresenta limitações, sendo necessários mais estudos para comprovar o seu benefício clínico. Para tal, é crucial entender a patogenicidade e os mecanismos de resistência de Mtb. A identificação de variantes genómicas ligadas à suscetibilidade diferencial aos β-lactâmicos é particularmente relevante para este objetivo. Um estudo recente revelou um grupo de marcadores genómicos putativos envolvidos na resposta aos β-lactâmicos, incluindo o gene lpqK (Rv0339c). Este gene codifica para uma lipoproteína putativa com um domínio β-lactamase. Além disso, os resultados obtidos mostraram que duas mutações num gene de hidrolase do PG, cwlM (Rv3915), estão associadas a menores concentrações mínimas inibitórias (CMIs) para os β-lactâmicos.
Para compreender a relação de cwlM e lpqK com um fenótipo de suscetibilidade aos β- lactâmicos, neste trabalho foram construídos mutantes de eliminação (knockdown) num modelo de estudo micobacteriano, o Mycolicibacterium smegmatis (M. smegmatis). Como Mtb é um patógeno humano que exige um laboratório de biossegurança de nível três e treino substancial antes do manuseio, traz consigo um risco de exposição acidental e a necessidade do uso de um organismo modelo não patogénico. Estes mutantes foram construídos usando o sistema clustered regularly interspaced short palindromic repeat (CRISPR) de interferência (CRISPRi). Este sistema foi otimizado por Rock et al. e é baseado no CRISPRi locus de Streptococcus thermophilus (dCas9Sth1). Para além disso, representa o método mais simples e rápido para regulação da transcrição de genes de interesse em micobactérias.
Com uma plataforma de plasmídeo único, tudo o que é necessário para o knockdown do gene é a clonagem de uma região de direcionamento única de ~20 pares de base do RNA guia (sgRNA). O
IV CRISPRi é indutível, permitindo assim a fácil manipulação de genes essenciais. A magnitude do knockdown de CRISPRi é ajustável, variando a "força" do motivo protoespaçador adjacente (PAM) direcionado, no caso da dCas9Sth1, ou variando a concentração de indutor, modulando o grau de complementaridade de sgRNA-alvo ou truncando a região de direcionamento do sgRNA. Finalmente, o CRISPRi pode ser multiplexado, o que é de particular importância em patógenos de crescimento lento, como Mtb em que a manipulação genética é demorada.
A viabilidade dos mutantes knockdown de cwlM e de lpqK em M. smegmatis foi verificada através de ensaios de diluição (spotting assays) e confirmamos a essencialidade de cwlM e a não essencialidade de lpqK em M. smegmatis. O knockdown foi posteriormente confirmado por PCR quantitativo em tempo real (qRT-PCR). A caracterização do fenótipo de suscetibilidade a antibióticos β-lactâmicos e anti-TB foi feita através de ensaios de CMIs. Nestes, conseguimos observar que a repressão de cwlM, afeta a suscetibilidade de M. smegmatis à cefotaxima e ao meropenem, no entanto, a repressão de lpqK não teve qualquer efeito na suscetibilidade aos vários antibióticos. Para além disso, foram feitos spotting assays com cefotaxima e meropenem, e discos de difusão em agar utilizando amoxicilina-clavulanato e meropenem. Estes ensaios foram realizados com apenas o mutante knockdown de cwlM uma vez que foi o único mutante no qual se registaram diferenças nos ensaios CMIs. A partir dos resultados obtidos, em conjunto, foi possível determinar que a repressão de cwlM tem influência no fenótipo de suscetibilidade de M. smegmatis em comparação com a estirpe wild-type, para a cefotaxima e para o meropenem.
Um outro objetivo deste projeto foi a caracterização das proteínas CwlM e LpqK de Mtb. A proteína CwlM está anotada com uma N-acetilmuramoil-L-alanina amidase, pertencendo ao grupo das hidrolases, que tem um papel de regulação numa via conservada relacionada com a biossíntese do PG:
PknB-CwlM-MurA. Após a indução, extração e purificação da CwlM, foi realizado um zimograma de forma a caracterizar uma possível atividade de hidrólise do PG, no entanto esta proteína não revelou atividade contra o PG de Micrococcus luteus. A proteína LpqK não foi purificada devido a constrangimentos no tempo de realização do projeto. A proteína LpqK é uma lipoproteína com um domínio de β-lactamase, como tal, esta atividade foi medida através de um ensaio de hidrólise de nitrocefina, em estirpes produtoras de ambas as proteínas ao invés de se testar a atividade das proteínas purificadas. Como resultado, foi possível verificar que LpqK tem a maior atividade de hidrólise em comparação com o controlo e que CwlM também tem alguma atividade embora menor que LpqK. Para perceber como a CwlM pode interagir com vários antibióticos em prol de um fenótipo de suscetibilidade/resistência de M. smegmatis, foram realizados ensaios de sinergia entre várias concentrações de CwlM com várias concentrações de cefotaxima, cefotaxima-clavulanato, meropenem e etambutol. Este ensaio revelou que a CwlM causa um atraso evidente no efeito bactericida da cefotaxima e em conjunto com o clavulanato há um efeito semelhante, mas mais reduzido. Esta observação é mais notória para a concentração de proteína mais elevada usada no ensaio em concentrações de antibiótico perto da CMI usual. Para os restantes antibióticos as diferenças não são notórias pois para qualquer concentração de antibióticos ou proteína, os valores de taxa de crescimento são bastante similares. Estes resultados podem dever-se à atividade de amidase que ClwM tem.
Usualmente, as β-lactamases agem de forma a cortarem a ligação amida nos β-lactâmicos e a CwlM poderá também estar a cortar essa ligação. Como cada antibiótico β-lactâmico tem uma estrutura diferente, a CwlM pode ter uma afinidade maior na quebra dessa ligação no caso da cefotaxima em comparação com os outros β-lactâmicos usados.
V Em conclusão, o trabalho apresentado nesta dissertação ajudou a esclarecer a importância do cwlM e do lpqK na suscetibilidade/resistência de M. smegmatis a antibióticos. Para além disso, foi possível perceber um pouco das características e da importância das proteínas ortólogas em Mtb para a resistência a antibióticos. Os resultados e as conclusões apresentadas poderão ser relevantes para uma melhor compreensão da patogénese de Mtb e contribuir para o desenvolvimento futuro de terapêuticas alternativas para o tratamento de TB. Estas terapêuticas alternativas poderão ser exploradas para amplificar a eficácia dos β-lactâmicos, através do uso de um adjuvante que iniba a ação de determinados alvos. Assim, será possível compreender a utilidade geral dos antibióticos β-lactâmicos na terapêutica da TB e promover a sua utilização contra isolados resistentes a antibióticos específicos.
Palavras-chave: Tuberculose; Resistência a antibióticos; CRISPRi; PG hidrolase; β-lactâmicos.
VI
Index
Acknowledgments... I Abstract... II Resumo... III Index...VI Figure Index... IX Table Index... XII Abbreviation List... XIII
1. Introduction... 1
1.1 Tuberculosis... 1
1.2 Mycobacteria... 2
1.2.1 Mycobacterial cell wall ... 2
1.3 Antibiotics targeting mycobacteria ... ... 4
1.3.1 The β-lactam antibiotics and their potential application in TB treatment ... 5
1.3.1.1 The molecular targets of the β-lactams... 5
1.3.1.2 β-lactams obstacles ... 5
1.3.1.3 β-lactam chemotherapy developments ... 6
1.4 Putative genomic markers involved in β-lactam susceptibility... 7
1.4.1 CwlM, a PG hydrolase... 7
1.4.2 LpqK, a lipoprotein ... 9
1.5 CRISPR ... 9
1.5.1 CRISPRi ... 11
1.5.1.1 Advantages ... 11
1.5.1.2 Disadvantages ... 12
1.5.2 CRISPRi in mycobacteria ... 12
2. Objectives... 14
VII
3. Materials and Methods... 14
3.1 Bacterial strains, plasmids, and culture conditions ... 14
3.2 Preparation of E. coli chemically competent cells (CCC) ... 15
3.3 Preparation of M. smegmatis electrocompetent cells (ECC) ... 15
3.4 Antibiotics and reagents stocks ... 16
3.4.1 ATc stocks ... 16
3.4.2 Antibiotic stocks for MIC, spotting assays and antibiotic disks assays ... 16
3.5 sgRNA design, plasmid cloning, and transformation ... 16
3.5.1 Amplification of the CRISPRi backbone ... 17
3.5.2 Cloning the sgRNA into the backbone vector ... 18
3.5.3 Selection of the sgRNA-containing CRISPRi plasmids ... 19
3.6 Evaluation of mRNA expression levels by qRT-PCR ... 19
3.7 Spotting assays ... 21
3.8 MICs ... 21
3.9 Antibiotic disks ... 21
3.10 Cloning of the cwlM and lpqk from Mtb in the pET-29b system ... 22
3.10.1 PCR amplification of the cwlM and lpqk genes from Mtb... 22
3.10.2 Restriction enzyme digestion of pET-29b vector and inserts... 23
3.10.3 Ligation of inserts to the pET-29b vector... 24
3.10.4 Transformation of E. coli JM109 with the ligation mixtures... 24
3.11 Optimization and production of the LpqK and CwlM proteins ... 25
3.11.1Transformation of E. coli BL21(DE3) competent cells with the pET-29b constructs for protein induction ... 25
3.11.2 Induction of the CwlM and LpqK proteins... 25
3.11.3 SDS-PAGE ... 25
3.11.4 Purification of E. coli BL21(DE3):cwlM... 25
VIII
3.12 Enzymatic Activity ... 26
3.12.1 Zymography ... 26
3.12.2 Nitrocefin ... 26
3.13 Antibiotic and protein synergy testing through the checkerboard method ... 27
4. Results and Discussion... 28
4.1 Construction and characterization of the cwlM and lpqK knockdown mutants in mycobacteria ... 28
4.1.1 Construction of the cwlM and lpqK knockdown mutants in M. smegmatis mc2155 using CRISPRi ... 29
4.1.2 Phenotypic characterization of the cwlM and lpqK knockdown mutants in M. smegmatis mc2155 ... 29
4.1.3 Assessment of CRISPRi-mediated gene repression by qRT-PCR ... 30
4.2 Antibiotics that target the mycobacterial CW biosynthesis ... 31
4.3 Cloning of cwlM and lpqk in the pET-29b system... 36
4.4 Characterization of the CwlM and LpqK proteins ... 38
4.4.1 Production of the CwlM and LpqK proteins ... 38
4.4.2 Purification of the CwlM protein ... 39
4.4.3 Enzymatic activity characterization ... 41
4.4.3.1 Zymogram ... 41
4.4.3.2 Nitrocefin hydrolysis assay ... 42
4.4.4 Synergy of the CwlM protein with antibiotics ... 43
5. Conclusions and Future Perspectives... 45
6. References... 48
7. Annexes... 52
IX
Figure Index
Figure 1.1 – The cell wall of Mycobacterium tuberculosis. MOM = mycobacterial outer membrane, LM = lipomannan, AM = arabinomannan, LAM = lipoarabinomannan, ManLAM = mannose-capped LAM, GlcNac = N-acetyl-glucosamine, MurNAc = N-acetyl-muramic acid, PDIM = phthiocerol dimycocerosate, TDM = trehalose dimycolate, PGL = Phenolic glycolipid. Retrieved from Raffetseder et al. (2019)………..………...3 Figure 1.2 – Biology of the type II-A CRISPR-Cas system. The type II-A system from S. pyogenes is shown as an example. (A) The cas gene operon with tracrRNA and the CRISPR array. (B) The natural pathway of antiviral defense involves association of Cas9 with the antirepeat-repeat RNA (tracrRNA:crRNA) duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target DNA cleavage. (C) Details of the natural DNAcleavage with the duplex tracrRNA:crRNA. Retrieved from Doudna et al. (2014) ………10 Figure 3.2 – Strategy used to design sgRNAs to repress cwlM using CRISPRi in mycobacteria. NT – non-template strand, T – template strand………..……….………...….17 Figure 3.12 – Synergy schematics between antibiotics and CwlM using the checkerboard method.……….………..28 Figure 4.1 – Spotting assays of M. smegmatis mc2155 WT, PLJR962, knockdown mutants (cwlM and lpqK mut) in the presence (+) and absence (–) of 100 ng mL-1 ATc. M. smegmatis WT and PLJR962 were used as negative controls. The PLJR962 mutant is a non-targeting control. The first spot corresponds approximately to 0.001 OD600 and each subsequent spot is a two-fold dilution. Each spot corresponds to the plating of 5 μL of culture volume. These data are representative of three independent experiments……….………...29 Figure 4.2 – Relative cwlM and lpqK mRNA expression levels in M. smegmatis mc2 155 WT, PLJR962 and knockdown mutants (cwlM and lpqK mut) with (striped bars) and without (monocolour bars) 100 ng mL-1 ATc treatment for 6 h. The left graph levels are for the cwlM gene, the right graph is for the lpqK gene. “ATc” after the name means the mutants were grown in the presence of ATc. M. smegmatis mc2 155 WT and PLJR962 were used as controls. The PLJR962 mutant is a non- targeting control. The data were quantified using the ΔΔCT method and normalized to the M. smegmatis housekeeping gene sigA, using M. smegmatis WT as a calibrator sample. The bars represent the mean of two independent experiments with two technical replicates each and the error bars correspond to the standard error of the mean (SEM). Asterisks indicate the significance of the difference in relative mRNA expression calculated using the Ordinary One-way ANOVA with Sidak’s multi comparisons test (* P <
0.05, ** P < 0.01, *** P < 0.001)……. ………..……….30 Figure 4.5 – Spotting assays of M. smegmatis mc2 155 and the cwlM knockdown mutant in presence of varying concentrations of cefotaxime and meropenem. A. negative control plates (no antibiotic);
B. Agar plates with cefotaxime; C. Agar plates with meropenem. Control – M. smegmatis WT. cwlM – cwlM knockdown mutant……….……..……….35 Figure 4.6 – Average length (cm) of the inhibition halo for amoxicillin (AMX+CLAV) and meropenem (MER) antibiotic disks for M. smegmatis mc2155 WT and for the cwlM knockdown
X mutant (mut), with (striped bars) and without (smooth bars) 100 ng mL-1 ATc. M. smegmatis mc2 155 WT was used as a negative control. Measurements were made by ImageJ software tools. The bars represent the mean of three independent experiments with three biological triplicates each and the error bars correspond to the standard deviation. Asterisks indicate the significance of the difference in relative diameter length calculated using the ordinary one-way ANOVA with Sidak's multiple comparisons test (* P < 0.05)……….……….36 Figure 4.7 – Agarose gels of the digested (XhoI and NdeI enzymes) and purified PCR products of the cwlM and lpqK genes of Mtb H37Rv and the pET-29b vector (A) and of pET-29b:cwlM construct (B). Panel A lane order (from left to right): ladder (GeneRuler 1 kb plus DNA Ladder - ThermoScientific); cwlM insert; lpqK insert; pET-29b vector. 100 ng µl -1 of the inserts were loaded into the lanes. Panel B lane order (from left to right): ladder (GeneRuler 1 kb plus DNA Ladder – ThermoScientific); negative clone (N) without the insert, with only the pET-29b vector showing;
positive clone (P) with the pET-29b:cwlM construct.
………...37 Figure 4.8 – SDS polyacrylamide gel of the supernatant of the non-induced and induced cell lysates of E. coli BL21 (DE3) expressing either CwlM or LpqK at different conditions. Order of the lanes from left to right: ladder (NZYColour Protein Marker II – NZYTech); E. coli BL21(DE3):pET- 29b:cwlM (non-induced (N.I); induced at 37ºC; induced at 16ºC); E. coli BL21(DE3):pET-29b:lpqK (non-induced; induced at 37ºC; induced at 16ºC). The red arrows indicate the presence of the proteins……….……….………..38 Figure 4.9 – SDS polyacrylamide gel of the supernatant of the non-induced and induced cell lysates of E. coli BL21 (DE3) expressing either CwlM or LpqK at 16 ºC O.N. Order of the lanes from left to right: ladder (NZYColour Protein Marker II – NZYTech); E. coli BL21(DE3):pET-29b:cwlM (non- induced (N.I); induced at 16ºC); E. coli BL21(DE3):pET-29b:lpqK (non-induced; induced at 16ºC).
The red arrows indicate the presence of the proteins……….………...39 Figure 4.10 – Absorbance at 280nm during the elution step of the affinity purification of CwlM from an induced filtrate of E. coli BL21(DE3):pET-29b:cwlM in the ÄKTAprime plus system. 1 through 11 are the fractions collected and the last dot is waste……….….……….….40 Figure 4.11 – Fractions of the CwlM protein after purification in the AKTA (A) and final purified protein (B). Panel image A order: ladder (NZYColour Protein Marker II – NZYTech), 3, 4, 5, 6, 7, 8, 9 and 10. Panel image B order: ladder (NZYColour Protein Marker II – NZYTech), CwlM………….……….40 Figure 4.12 – SDS-PAGE (A), Zymogram (B) and plate (C) of Micrococcus luteus with the CwlM protein. Lane order of both gels: CwlM, ladder, BSA (negative control), Lysozyme (Lys – positive control)………42 Figure 4.13 – Nitrocefin assay with activity expressed as µg of hydrolysed nitrocefin min-1 by mg of total protein-1 for each protein (pET-29b – control, CwlM and LpqK). Ordinary one-way ANOVA Dunnett's multiple comparisons test was applied for relative statistical differences………….43
XI Figure 4.14 – Synergy graphs with M. smegmatis mc2 155 growth rate by antibiotic concentration (µg ml-1) for each CwlM protein concentration (µg ml-1)………44 Figure 4.15 – Scaffold of β-lactam antibiotics (cefotaxime and meropenem) and ethambutol. The amide bond in the β-lactam ring is highlighted in red……….….45 Figure 7.1 – The CRISPRi-dCas9Sth1 system optimized by Rock et al. in mycobacteria. (A) CRISPRi backbone plasmids for M. smegmatis (PLJR962) with indication of enzymatic restriction sites.
bp, base pair; dCas9, deficient Cas9 protein; KANR, kanamycin resistance cassette; L5 int, single copy L5-inegrative backbone with the integrase gene and an attP site; OriE, E. coli derived pBR3222 origin of replication; Sth1 dCas9, dCas9 from S. thermophilus. (B) Different PAM position variants for dCas9Sth1. The 15 PAMs (PAM1-15) that were described as able to cause a >25-fold repression are indicated by a blue rectangle. PAM, proto-spacer adjacent motif; SD, standard deviation; sgRNA, single guide RNA. Adapted from Rock et al. (2017)...52 Figure 7.2 – Constructs of pET-29b:cwlM and pET-29b:lpqK represented by Snapgene software.
A – pET-29b:cwlM; B – pET-29b:lpqK...54 Figure 7.3 – NCBI BLASTp analysis: Rv3915 alignment with MSMEG_6935...57 Figure 7.4 – NCBI BLASTp analysis of the Rv0399c amino acid sequence against the M. smegmatis mc2 155 genome (taxid:246196), produces a significant alignment with the MSMEG_4455...58 Figure 7.5 – NCBI BLASTp of Rv0399c (A) and MSMEG_4455 (B) against multiple data bases.
Top 10 results are showed………...59 Figure 7.6 – Result of MotifFinder of the CwlM protein against multiple data bases.
(https://www.genome.jp/tools/motif/)...60 Figure 7.7 – Result of MotifFinder of the LpqK protein against multiple data bases.
(https://www.genome.jp/tools/motif/)...61
XII
Table Index
Table 3.1 – Bacterial Strains and Plasmids used in this study……….………...15 Table 3.2 – sgRNAs used to target cwlM and lpqK genes in M. smegmatis……….17 Table 3.3 – Primers used to clone the cwlM and lpqK targeting sgRNAs into PLJR962. Fwd – forward, Rv – reverse.……….………18 Table 3.4 - Primers used to quantify the expression of cwlM and lpqK in M. smegmatis by qRT- PCR………20 Table 3.5 – PCR primers used to amplify and create compatible inserts for cloning into the pET- 29b system. Fwd – forward, Rv – reverse.………..22 Table 3.6 – PCR reagents used in the amplification of the cwlM and lpqK genes………..22 Table 3.7 – PCR programs used in the amplification of the cwlM and lpqK genes………23 Table 3.8 – Concentrations of the backbone plasmid and PCR products after purification with A260/A280 and A260/A230 ratios.………...23 Table 3.9 – Concentrations of the digested products after purification with A260/A280 and A260/A230 ratios.………..24 Table 3.10 – Concentrations of the extracted plasmid DNA from the transformants with A260/A280 and A260/A230 ratios.………...24 Table 4.1 – EUCAST non-species related PK-PD breakpoints for β-lactams (μg mL-1). S – susceptible; R – resistant……….31 Table 4.2 – Median of Minimum Inhibitory concentrations for each strain of interest. M. smegmatis WT and PLJR962 are used as controls. Knockdown mutants (cwlM and lpqK mut). CLA – clavulanate;
AMX– amoxicillin; CTX – cefotaxime; MER – meropenem; EMB – ethambutol; INH – isoniazid; VAN – vancomycin. Presence of ATc (+), Absence of ATc (-)………32 Table 7.1 – Sequences of the primers used for the sequencing of positive clones with pET- 29b:cwlM and pET-29b:lpqK constructs. Fwd – forward, Rv – reverse...55
XIII
Abbreviation List
AG – Arabinogalactan
AIDS – Acquired immune deficiency syndrome AM – arabinomannan
AMX – Amoxicillin
AMX+CLAV – Amoxicillin combined with clavulanate ATc – Anhydrotetracycline
bp – Base pair
CCC - Chemical Competent cells CFUs – Colony forming units CLAV – Clavulanate
CRISPR – Clustered regularly interspaced short palindromic repeat CRISPRi – CRISPR interference
crRNA – CRISPR RNA CTX – Cefotaxime
CTX+CLAV – Cefotaxime combined with clavulanate CW – Cell wall
D-Ala-D-Ala – D-Alanyl-D-Alanine
D-Ala-mDAP – D-alanine – meso-diaminopimelic acid D-GalN – Galactosamine moietie
ECC – Electrocompetent cells EMB – Ethambutol
Fw – Forward
Galf – Galactose-furanose GlcNAc – N-acetyl-glucosamine HIV – Human immunodeficiency virus
XIV INH – Isoniazid
LAM – Lipoarabionomannan LB – Luria-Bertani
LDTs – L,D-transpeptidases LM – Lipomannan
MA – Mycolic acids
mAGP – Mycolyl-arabinogalactan-peptidoglycan ManLAM – mannose-capped LAM
mDAP – Meso-diaminopimelic acid MDR – Multi-drug resistant
MER – Meropenem
MER+CLAV – Meropenem combined with clavulanate MIC – Minimum inhibitory concentration
MOM – Mycobacterial outer membrane MTBC – Mycobacterium tuberculosis complex MurNAc – N-acetyl-muramic acid
MurNGly – N-glycolylmuramic acid nt – Nucleotides
NT strand – Non-template strand NTM – Non-tuberculous mycobacteria ON – Overnight
ORF – Open reading frame
PAM – Proto-spacer adjacent motif PBPs – Penicillin binding proteins PBS – Phosphate buffer saline PCR – Polymerase chain reaction
XV PDIM – Phthiocerol dimycocerosate
PG – Peptidoglycan PGL – Phenolic glycolipid
PK/PD – Pharmacokinetic/pharmacodynamic pre-crRNA – Long primary CRISPR RNA pre-XDR-TB – Pre-extensively drug-resistant TB
qRT-PCR – Quantitative real-time polymerase chain reaction RF – Rifampicin
rpm – Rotations per minute
RR-TB – Rifampicin-resistant tuberculosis Rv – Reverse
SEM – Standard error of the mean sgRNA – Single guide RNA T strand – Template strand TB – Tuberculosis
TDM – trehalose dimycolate
trans-crRNA – trans-acting CRISPR RNA TSS – Transcription start site
U – Units
UTR – Untranslated region VAN – Vancomycin
WHO – World Health Organization WT – Wild-type
XDR – Extensive-drug resistant
1
1. Introduction
1.1
Tuberculosis
Tuberculosis (TB) is a lethal infectious disease, generally affecting the lungs (pulmonary TB), while other organs can still be affected (extrapulmonary TB). Since ancient times, it has been a constant threat to humanity and it may have killed more people than any other microbial pathogen. The bacterium that causes TB in humans, Mycobacterium tuberculosis (Mtb), was cultured and identified for the first time by Robert Koch in 18821. In the 1940s, Selman Waksman discovered the first antibiotic active against Mtb, streptomycin, isolated from Streptomyces griseus. At that time, many considered that TB would soon be eradicated, an idea which was further supported by the discovery of new antibiotics2. However, drug resistance (DR) was quick to appear, and the challenges of the contemporary endemic TB have evolved into multi drug-resistant (MDR) strains of Mtb and co-infections with other diseases such as human immunodeficiency virus (HIV)3. According to the latest annual report on TB by the World Health Organization (WHO), in 2020, a total of 1.3 million people died from TB in that year (including 214 000 people with HIV) and an estimated 10 million people fell ill with TB worldwide. TB is the 13th leading cause of death and the second leading caused by a single infectious agent after COVID-19 (above HIV/AIDS). Between 2018–2020 only 50% and 32% of the TB and MDR/
rifampicin-resistant TB (RR-TB) cases, respectively, were successfully treated. In 2020, 86% of new TB cases occurred in the 30 high TB burden countries. Eight low-income overpopulated countries accounted for two thirds of the new TB cases: India, China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa. Additionally, Portugal still is the western european country with the highest incidence rate of TB, with an incidence rate of 14.2 per 100 000 inhabitants in 20204. Another problem is the existence of a vast reservoir of latent TB, in which about a quarter of the world’s population is latently infected with Mtb and 5 to 10% will develop TB disease during their lifetime5.
Usually, active TB and drug-susceptible Mtb is treated with a standard six-month course of four antimicrobial drugs (isoniazid, pyrazinamide, ethambutol, rifampicin). DR emerges when anti-TB medicines are used inappropriately, through incorrect prescription by health care providers, poor quality drugs, disruption of supply chains, and poor patient adherence. WHO uses five categories to classify cases of drug-resistant TB: isoniazid-resistant TB, RR-TB, MDR-TB, pre-extensively drug-resistant TB (pre-XDR-TB) and XDR-TB. MDR-TB is a form of TB caused by bacteria that do not respond to isoniazid and rifampicin, the two most effective first-line anti-TB drugs. MDR-TB is treatable and curable by using second-line drugs. However, second-line treatment options are limited and require extensive chemotherapy (up to two years of treatment) with medicines that are expensive and toxic. Pre- XDR-TB is TB that is resistant to rifampicin and any fluoroquinolone (a class of second-line anti-TB drug). XDR-TB is TB that is resistant to rifampicin, to any fluoroquinolone, and to bedaquiline or linezolid. This brings another problem, TB caused by bacteria that do not respond to the most effective second-line anti-TB drugs can leave patients without any further treatment options5.
To mitigate these problems, investments in TB prevention, diagnosis, treatment and research are needed with a global commitment6.
2 1.2
Mycobacteria
Mycobacteria belong to the order Actinomycetales, and most mycobacteria belong to the non- tuberculous large group often found in the soil. These can cause opportunistic infections but are usually non-pathogenic7,8. Traditionally, Mycobacterium species, the only genus in the family Mycobacteriaceae, have been divided into fast-growers and slow-growers, with the three major mycobacterial pathogens of humans: the Mycobacterium tuberculosis complex (MTBC), Mycobacterium leprae and Mycobacterium ulcerans, belonging to the slow-growers group. The MTBC includes organisms that can cause tubercular human disease, one of which is Mtb9.
As described above, based on growth rate, mycobacteria are conventionally divided into two groups: the slow growers require more than one week to develop visible colonies on solid media, while the rapid growers may require three to seven days, thus growing slower in comparison with other cultivable bacteria10.
Mtb is a slender rod-shaped bacterium with a typical size of 0.2–0.6 (width) × 1–10 µm (length)11. Mtb is an intracellular pathogen, non-motile, aerobic to facultatively anaerobic, and has been described as non-spore forming and having a peripheral capsule8,11,12. Additionally, it has been demonstrated that Mtb divides asymmetrically, as opposed to the symmetric cell division documented for many other bacteria, producing two daughter cells of different sizes13.
Mtb is a Category Three human pathogen, requiring handling in a dedicated biosafety level three laboratory, substantial training prior to handling, and carrying with it a risk of accidental exposure10. Mtb fits in the first group of the slow growers, doubling every 18-24 h in liquid culture. Thus, colony formation requires three to four weeks, making each experiment time consuming. Mycolicibacterium smegmatis (M. smegmatis) is a soil dwelling saprophytic mycobacterial species that is a distant relative of Mtb. This avirulent mycobacterial species is fast growing, with a doubling time of approximately three hours and colony generation in two to three days. Therefore, it is a common surrogate for Mtb in research10,11,14.
1.2.1 Mycobacterial cell wall
Mtb is a GC-rich gram-positive-like bacteria that forms a monophyletic group with the low GC- rich gram-positive bacteria such as Bacillus subtilis, although its cell wall (CW) features an outer membrane as in gram-negative bacteria15. Due to its waxy, impermeable CW, the Gram staining technique often renders inconsistent results. Instead, mycobacteria can be identified by a procedure known as acid-fast staining8,16.
The Mtb cell envelope is a complex structure. The inner membrane phospholipid bilayer contains glycolipids that extend into the periplasmic space. The essential core CW structure is composed of three main components: a cross-linked polymer of peptidoglycan (PG), a highly branched arabinogalactan (AG) polysaccharide, and long-chain mycolic acids (MA). Intercalated into the mycolate layer are solvent-extractable lipids, including non-covalently linked glycophospholipids and inert waxes, forming the outer membrane. The capsule forms the outermost layer and is mainly composed of proteins and polysaccharides (α-glucans and arabinans)12,17.
3
Figure 1.1 – The cell wall of Mycobacterium tuberculosis. MOM = mycobacterial outer membrane, LM = lipomannan, AM
= arabinomannan, LAM = lipoarabinomannan, ManLAM = mannose-capped LAM, GlcNac = N-acetyl-glucosamine, MurNAc
= N-acetyl-muramic acid, PDIM = phthiocerol dimycocerosate, TDM = trehalose dimycolate, PGL = Phenolic glycolipid.
Retrieved from Raffetseder et al. (2019) 18.
PG is a major component of the CW of both gram-positive and gram-negative bacteria. It is a polymer of alternating N-acetylglucosamine (GlcNac) and N-acetylmuramic (MurNAc) acid residues via β(1→4) linkages with side chains of amino acids cross-linked by transpeptide bridges. PG has a multitude of purposes in addition to giving shape and stiffness. It also counteracts turgor pressure, making it crucial for growth and survival. Since PG is specific to bacterial cells, it has been the target of numerous potent antibiotics, specifically the enzymes involved in its synthesis are promising candidates for future antibiotic research17.
The mycobacterial PG has several unique features that differentiate it from the typical structure, including a mixture of the MurNAc and N-glycolylmuramic (MurNGlyc) acid derivatives, the amidation of D-iso-glutamate, the amidation of m-DAP carboxylic acids and, the occurrence of complex side chains (by addition of glycine or serine residues). The mycobacterial PG is also heavily cross-linked, through the action of D,D-transpeptidades (PBPs) catalysing the formation of 4→3 (D-Ala-mDAP) bonds or the action of non-classical L,D-transpeptidases (LDTs) catalysing the formation of 3→3 (mDAP-mDAP) bonds. The degree and type of cross-linking contributes to the increased stability and complexity of the CW17.
The major CW polysaccharide, AG, as the name suggests, is composed of galactose (Gal) and arabinose sugar residues, in the furanose (f) ring form (Galf). AG is attached to PG via a single linker unit, covalently bound. The galactan component is a linear chain of approximately 30 alternating five- and six-linked β-D-Galf residues. Three highly branched arabinan chains, consisting of approximately 30 Araf residues, are attached to the galactan chain. The non-reducing termini of the arabinan chains act as an attachment site for the MA, succinyl and galactosamine (D-GalN) moieties17.
4 The Mtb CW contains an outer layer of unique MA that are C60–C90 long chain carboxylic acids covalently linked to AG19,20. These unique long chain α-alkyl-β-hydroxy fatty acids (comprised of a main meromycolate chain of C42–C62 and a long saturated α-chain C24–C26) also make up other outer cell envelope lipids17. The MA are generally perpendicular to the surface of the cell but their boundary with the hydrophilic extracellular milieu or capsular area is mediated by a variety of accessory lipids, such as trehalose mono/di-mycolates and glucose monomycolate, phthiocerol dimycocerosate, sulfolipids, and glycolipids, including lipomannan and lipoarabinomannan. The MA on the inner leaflet and the free glycolipids on the outer leaflet effectively form a mycobacterial outer membrane bilayer19.
Together, the MA, the AG and the PG compose the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex, a crucial macromolecule that encases the cell19. Being the main feature of the mycobacterial cell envelope and an intricate structure, the mAGP complex is responsible for the innate resistance to many commonly used antibiotics and is also involved in virulence19,20.
1.3
Antibiotics targeting mycobacteria
The lipid- and carbohydrate-rich layers of the CW serve not only as a permeability barrier, providing protection against hydrophilic compounds, but are also critical in pathogenesis and survival17. Enzymes that catalyse biosynthesis and recycling of CW components, in particular the PG, are essential to the viability of Mtb. Therefore, these are attractive targets for novel antibiotics research20.
Standard TB treatment for drug-susceptible disease requires the simultaneous use of four drugs:
rifampicin, an RNA transcription inhibitor; isoniazid, a MA biosynthesis inhibitor; ethambutol, an AG biosynthesis inhibitor; and pyrazinamide, which inhibits the recycling of stalled ribosomes5,21. The side effects of the therapeutic regimen can be severe and may discourage many patients from completing the course of treatment. Furthermore, the six-month duration of the treatment makes ensuring its completion difficult in impoverished areas with poor public health systems. Infection with MDR-TB and XDR-TB requires treatment with various second-line antibiotics that are expensive, have far more side effects due to their higher toxicity, and are part of therapeutic regimens of longer duration5. The only newly approved drugs for TB over the past 50 years are the second-line drugs for treating MDR-TB: linezolid, pretomanid, bedaquiline and delamanid, but not long after their introduction in TB therapy, resistance emerged19, 21–23.
Numerous members of the antibiotic classes widely used to treat respiratory tract infections, such as β-lactams, fluoroquinolones, and macrolides, are notably absent from this drug list19. Fluoroquinolones are nucleic acid synthesis inhibitors used in second-line therapy and newer fluoroquinolones are currently in phase III clinical trials as first-line agents21,24. Although macrolides, which are protein synthesis inhibitors, are effective against other mycobacteria, Mtb is inherently resistant to this class of antibiotics. For resistant Mtb, other widely used drug classes such aminoglycosides (like streptomycin) may also be employed21. However, β-lactams have not yet been widely used in clinical settings to treat TB19. Several of these drugs are being selected for possible repurposing applications, because they have several advantageous characteristics like being affordable, easily available, and possessing favourable pharmacokinetic/pharmacodynamic (PK/PD) properties19,22.
5 1.3.1 The β-lactam antibiotics and their potential application in TB treatment
Since β-lactams are a bactericidal class of antibiotics with well-characterized PK/PD properties, they are among the oldest and best-validated antibiotics in medical practice. They are the chosen treatment for a wide range of infections, both minor and serious ones, brought on by various bacterial pathogens19,22, and their exclusion from Mtb treatment is notable.
Through a variety of mechanisms, including inactivation by β-lactamases, modification of the target protein in the CW, and extrusion from the periplasmic space by efflux pumps, pathogens have developed resistance in multiple environments25. However, strategies have been developed to overcome some of these resistance mechanisms in other clinical situations, in part due to their incredible effectiveness against susceptible organisms. The ability to use β-lactams against some organisms with β-lactamases or resistant PBPs has been made possible by the modification of β-lactams to evade β- lactamase activity, concurrent therapy with β-lactamase inhibitors, and optimization of β-lactams to target the PBPs of particular pathogens19.
Recently, the rise of MDR-TB has encouraged renewed interest in β-lactam/β-lactamase inhibitor combination therapy19,26.
1.3.1.1 The molecular targets of the β-lactams
Exposure to β-lactams may induce several different effects. The observable outcomes range from bacteriostasis and morphological alterations to rapid bacteriolysis. All known effects are caused by the inhibition of PG-modifying peptidases outside of the cell membrane, despite effects varying by species, antibiotics, and exposure circumstances. These peptidases are the PBPs and the LDTs. In short, inside the cell the β-lactams mimic the natural D-Ala-D-Ala substrate of the PBPs and inhibit them by forming a slowly hydrolysing adduct at the active site19,25. There are, however, others that act on the PG precursors. For example, the glycopeptides, vancomycin and teicoplanin, bind to the D-Ala-D-Ala terminus of the pentapeptide stem, preventing polymerization reactions17.
The two most diverse classes of β-lactams, the penicillins and cephalosporins, cause bacteriolysis and cell death that probably occurs through the inhibition of the high molecular weight (HMW) PBPs in detriment of the LDTs19,25. This happens because LDTs are resistant to most β-lactam antibiotics, except the carbapenems17.
The third major class of β-lactams, the carbapenems, also display high affinity for certain Class A HMW PBPs, and so it has been presumed that the carbapenems target these PBPs too. The finding that carbapenems form adducts with the LDTs, and that they potently kill organisms rich in these cross- links, has established a great advantage for this antibiotic class with LDTs being an additional or alternate molecular target for the carbapenems19,27.
1.3.1.2 β-lactams obstacles
Two obstacles hinder β-lactams during their passage through the CW to their transpeptidase targets in the Mtb PG. They must first pass the hydrophobic, poorly permeable MA layer which makes up a significant portion of the CW. Then, they must escape the hydrolytic activity of Mtb’s promiscuous, highly active β-lactamase, BlaC19.
6 Several properties of the MA make them formidable obstacles to the passage of β-lactams and other small molecules. These include their size or length, their structure and their unusual modifications or unsaturations of the alkyl chain, apart from the hydrophobicity of the MA. The different types of unsaturation, which may vary across species, likely contribute to the higher or lower permeability of the cell walls of different mycobacteria19.
Beyond the outer membrane, β-lactams enjoy unhindered passage through the hydrophilic AG to the PG target area. The properties of the PG, such as glycan chain length and the type and density of cross-linking, are the product of the PG synthetic machinery that is the target of the β-lactams. These properties may reflect the relative importance of individual components of the PG synthetic machinery and determine which ones are viable antibiotic targets. As with the glycan chain length, the degree of peptide cross-linking modulates the properties of the PG macromolecule. More cross-linking results in a tighter mesh encircling the cell. Further than the degree of cross-linking, the nature of the cross-link also appears to have a major impact on bacterial physiology. Additionally, a large proportion of the CW PG is cross-linked by LDTs, which are intrinsically impervious to these antibiotics19,20.
This unusual cross-linking is just one of several properties that contribute to the complexity of the mycobacterial cell envelope as a β-lactam resistance mechanism. Combined with the low permeability of the MA layer and the mycobacterial β-lactamase, these represent an array of difficulties that β-lactam chemotherapy must overcome19.
Beyond the cross-linking, two broad classes of resistance mechanisms have occurred repeatedly.
The first encompasses mechanisms that decrease the effective concentration of the β-lactams at the general site of action, the periplasm, or the CW zone. This class comprises changes in outer membrane permeability, β-lactamase activity and efflux pump activity19, mainly based on intrinsic resistance28. The second broad class of β-lactam resistance mechanisms is the modification of the PBP profile of the cell, in such a way that sufficient transpeptidase activity remains to permit survival, even in the presence of the β-lactam. This may occur through target site modification, in which an individual PBP acquires a mutation that alters its affinity for the β-lactams19,28, or through acquisition or activation of previously unused PBPs with low β-lactam affinity19. Acquired DR in mycobacterial species, is caused mainly by spontaneous mutations in chromosomal genes encoding drug targets28.
In conclusion, Mtb is resistant to the majority of antimicrobial classes as a result of the unique characteristics of the CW, the accumulation of chromosomal mutations, and drug degradation/modification caused by the presence of antibiotic inactivating enzymes28. However, the inhibition of PG synthesis by potent transpeptidase inhibitors, such as carbapenems or glycopeptides, protected by drugs that block β-lactamase activity, and in combination with other CW inhibitors, may result in enhanced efficacy of these antibiotics20.
1.3.1.3 β-lactam chemotherapy developments
Until recently, β-lactams were not considered for use in the treatment of TB, mostly due to the expression of the broad-spectrum β-lactamase, BlaC. However, it has been demonstrated that BlaC hydrolyses carbapenems at a low rate and is permanently inactivated by clavunalate29. Combinations of β-lactam and β-lactamase inhibitor therapy are bactericidal against both replicating and non-replicating forms of Mtb, and clinical trials are currently being conducted to test these combinations17,26. Tebipenem/clavulanate and meropenem/clavulanate have shown potent efficacy against drug-resistant
7 Mtb, despite the fact that amoxicillin/clavulanate had only limited success in multiple trials. This finding has led to a successful animal trial and preliminary clinical trials of meropenem/clavulanate against MDR/XDR-TB, resulting in significant improvements in outcomes for treated patients versus controls19,
29,30. Though meropenem must be administered intravenously, tebipenem is an oral carbapenem and may be a better option in developing new anti-TB regimens26. These results are encouraging because β- lactams inhibit a biosynthetic pathway that is not targeted by current treatments, and resistance to those treatments should have little to no effect on β-lactam therapy19.
1.4 Putative genomic markers involved in β-lactam susceptibility
The application of β-lactams still has certain limitations and further studies are necessary to identify cases in which the addition of a β-lactam to the therapeutic regimen may result in a greater clinical benefit. The identification of genomic variants linked with increased or decreased susceptibility to β-lactams is particularly relevant for this goal.
A study conducted in our laboratory, which combined whole-genome sequencing and β-lactam susceptibility screening of a total of 172 Mtb clinical isolates obtained from the National Institute of Health Doutor Ricardo Jorge, revealed a group of 20 putative genomic markers of differential β-lactam phenotypes, where the lpqK (Rv0339c) gene is included31. In addition, the obtained results showed that strains with more than two mutations in PG hydrolase genes had lower minimum inhibitory concentrations (MICs) to amoxicillin and meropenem, both with and without clavulanate. Among the most common mutations in these targets we find the M237V substitution in cwlM (Rv3915), which was associated with lower β-lactam MICs when compared to the values obtained for the global sample31. Therefore, better characterization of these two target genes and their potential role within the mechanisms of action and resistance of β-lactams is important.
1.4.1 CwlM, a PG hydrolase
The PG layer provides protection and shape-defining structure to cells of almost all bacterial species. This layer is constructed by transpeptidases and glycosyltransferases, which attach new precursors to the existing CW and several types of catabolic PG hydrolases/autolysins, which break bonds in the existing PG32. These enzymes, which are classified into glycosidases (including muramidases and glucosaminidases), amidases and peptidases, have been proposed to have roles in bacterial surface growth, cell division and bacterial pathogenesis, including adhesion and invasion of host cells33.
This activity is both essential and potentially harmful to the organism, and the expression, distribution, and activation of PG enzymes are likely to be tightly controlled, including translational, transcriptional and post-transcriptional regulation in order to promote cell growth and septation without compromising the CW integrity32,33. Accordingly, many regulators in the cytoplasm, inner membrane and periplasm, coordinate and control the activities of PG enzymes, either directly or indirectly. In addition to the dedicated regulators, many PG synthases and hydrolases work together in complexes and regulate each other’s enzymatic activity through protein-protein interactions32.
8 The cwlM gene is predicted to be essential for growth in Mtb, is highly conserved among mycobacteria, and is annotated as encoding an N-acetylmuramoyl-L-alanine amidase of the AmiA/LytC family, hereafter referred to as a PG amidase, a type of PG hydrolase32.
In an analysis of the Mtb genome, Deng et al. identified an open reading frame (ORF) with homology to the PG hydrolase gene cwlB of Bacillus subtilis. Although the encoded protein, CwlM, possesses two putative PG binding domains in its sequence, it is a soluble protein and, unexpectedly, lacks a trans-membrane domain and the typical signal peptide33. In contrast, Boutte et al., in studies with cwlM and the correspondent orthologue gene in M. smegmatis (MSMEG_6935), found that the essential function of the predicted PG hydrolase is regulatory rather than enzymatic. These authors also found that CwlM is phosphorylated by the essential serine threonine protein kinase (STPK) PknB in the cytoplasm, and functions to stimulate the catalytic activity of MurA, the first enzyme in the PG precursor synthesis pathway. In nutrient-replete conditions, CwlM is phosphorylated (CwlM~P) and increases the rate of MurA catalysis by ~30 fold. In starvation, CwlM is dephosphorylated and does not activate MurA, which has very low activity alone32. Normally, overexpression of highly active PG hydrolases usually results in cell lysis but the overexpression of CwlM did not affect cell viability. This supports the hypothesis that the function of CwlM is regulatory rather than enzymatic32.
Boutte et al. found that CwlM-depleted cells fail to elongate normally and this could be due to uncoordinated synthesis of the various layers of the CW. Since PG biosynthesis is downregulated but other CW biosynthetic and metabolic processes may remain active, this leads to a CW with poor structural integrity32.
In short, Turapoc et al. proposes that growing Mtb produces two forms of CwlM: a non- phosphorylated membrane-associated CwlM and a PknB-phosphorylated cytoplasmic CwlM. The phosphorylated CwlM binds to FhaA, a fork head-associated domain protein, while non-phosphorylated CwlM interacts with MurJ (MviN), a proposed lipid II flippase. With this, CwlM potentially regulates both the biosynthesis of PG precursors and their transport across the cytoplasmic membrane. These results imply that a balance needs to be maintained between the phosphorylated and non-phosphorylated forms of CwlM, and that this fine balance is essential for bacterial viability and can be affected by altered PknB expression or activity34.
Decreased synthesis of PG precursors and CW metabolism is an important adaptation to stress.
Many models suggest that decreased metabolism contributes to the antibiotic tolerance of Mtb during treatment. This is based on the observation that many antibiotics are less effective during starvation and the stationary phase of growth in Mtb and other bacteria, likely because the activity of drug targets is reduced, or because permeability is decreased32,35. The PknB-CwlM-MurA signaling cascade functions to quickly turn off PG synthesis during nutrient restriction, and this regulation contributes to antibiotic tolerance during growth and under changing conditions32.
The ubiquitousness of these proteins among mycobacteria suggests that the PknB-CwlM-MurA regulatory pathway is conserved, although it is likely that different stresses activate the regulation of MurA in Mtb compared to M. smegmatis. Understanding this system could provide unique insights into the pathogen and facilitate the development of new treatments that interfere with the regulation of the mycobacterial CW during infection. This could shorten treatment times and improve patient outcomes32,33.
9 1.4.2 LpqK, a lipoprotein
The lpqK gene is an non-essential gene for the in vitro growth of Mtb (strain ATCC 25618/H37Rv), encoding a possible conserved lipoprotein31,36,37. This conserved lipoprotein shows some similarity to PBPs and various peptidases. LpqK is also similar to other PBPs and esterases of Mtb, and contains a predicted N-terminal signal sequence and a β-lactamase domain, meaning it could be involved in CW and cell processes31,37. In M. smegmatis, the orthologue of lpqK (MSMEG_4455) also has a N-terminal signal sequence and a β-lactamase domain.
The design and characterization of knockdown mutants of the cwlM and lpqK orthologue genes in M. smegmatis (MSMEG_6935 and MSMEG_4455, respectively), obtained by a precise transcription regulation tool (CRISPRi), can contribute to a better understanding of the role of the encoded proteins and anticipate the outputs of studies with Mtb.
1.5 CRISPR
Bacteria and archaea have evolved RNA-mediated adaptive defined systems called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with endoribonucleases (Cas) that protect organisms from invading viruses and plasmids. These defence systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids38. Functional CRISPR-Cas loci comprise a CRISPR array of identical repeats intercalated with invader DNA-targeting spacers that encode the CRISPR RNA (crRNA) components and an operon of cas genes encoding the Cas protein components39.
The CRISPR-Cas mediated prokaryotic immune defence occurs in three steps. The first step is adaptation, in which new spacers are acquired from integrating short fragments of foreign sequences into the host’s chromosome at the proximal end of the CRISPR array38–41. In the expression and interference phases, occurs the transcription of the repeat-spacer element into precursor CRISPR RNA (pre-crRNA) molecules followed by enzymatic cleavage liberating small interfering crRNAs that can base pair with complementary protospacer sequences of exogenous nucleic acid targets. Target recognition is the final step, in which crRNAs act as template guides directing the cleavage of the homologous/foreign sequences (protospacer) by means of nuclease Cas proteins that function in complex with the crRNAs38–40.
CRISPR-Cas systems can be divided into three types depending on the cas gene content and mechanism of immunity. In type II systems, the biogenesis of small crRNAs requires the coordinated action of the Cas9 nuclease, the host RNase III and a small transactivating crRNA (tracrRNA)42. The trans-activating tracrRNA is, thus, a small non-coding RNA with two critical functions: triggering pre- crRNA processing by RNase III and subsequently activating crRNA-guided DNA cleavage by Cas938. This system is found in Streptococcus thermophilus and Streptococcus pyogenes38,39.
10 Cas9 is a crRNA-guided double-stranded DNA endonuclease with two domains, RuvC and HNH, each of which cleaves one strand within the target DNA. In addition to the match between the guide crRNA and the protospacer sequence, Cas9 also requires the presence of a typically two–five nt (nucleotides) highly conserved sequence motif downstream of the protospacer sequence motifs, known as the protospacer-adjacent motif (PAM). After crRNA processing, this enzyme is loaded with a crRNA guide containing 20 nt of the spacer sequence followed by 19–22 nt of repeat sequence. Target recognition and cleavage require a match between the target and a ‘seed’ sequence of 12–15 nt at the 3’
end of the guide sequence, as well as a PAM38–40,42 .
Figure 1.2 – Biology of the type II-A CRISPR-Cas system. The type II-A system from S. pyogenes is shown as an example.
(A) The cas gene operon with tracrRNA and the CRISPR array. (B) The natural pathway of antiviral defence involves association of Cas9 with the antirepeat-repeat RNA (tracrRNA:crRNA) duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target DNA cleavage. (C) Details of the natural DNAcleavage with the duplex tracrRNA:crRNA. Retrieved from Doudna et al. (2014)39.
Through the creation of systems that use innately gene-regulation mechanisms to regulate gene expression, recombinant DNA technologies have revolutionized the study of gene function. Over the past ten years, the relative ease with which Transcription Activator-Like Effectors (TALENs) and Zinc Finger DNA-binding proteins (ZFN) have been reprogrammed to bind particular sequences has been utilized to control transcription activation or repression. Nonetheless, sequence specificity engineering for these proteins is still a time- and money-consuming procedure42. However, the field of biology is now experiencing a transformative phase with the advent of facile genome engineering in both eukaryotes and prokaryotes using RNA-programmable CRISPR-Cas9, originating from type II CRISPR-Cas systems39,42.
