Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosis

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In Mtb-infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1β secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.info:eu-repo/semantics/publishedVersio

Identification of drivers of mycobacterial resistance to peptidoglycan synthesis inhibitors

Beta-lactams have been excluded from tuberculosis therapy due to the intrinsic resistance of Mycobacterium tuberculosis (Mtb) to this antibiotic class, usually attributed to a potent beta-lactamase, BlaC, and to an unusually complex cell wall. In this pathogen, the peptidoglycan is cross-linked by penicillin-binding proteins (PBPs) and L,D-transpeptidases, the latter resistant to inhibition by most beta-lactams. However, recent studies have shown encouraging results of beta-lactam/beta-lactamase inhibitor combinations in clinical strains. Additional research on the mechanisms of action and resistance to these antibiotics and other inhibitors of peptidoglycan synthesis, such as the glycopeptides, is crucial to ascertain their place in alternative regimens against drug-resistant strains. Within this scope, we applied selective pressure to generate mutants resistant to amoxicillin, meropenem or vancomycin in Mtb H37Rv or Mycolicibacterium smegmatis (Msm) mc2-155. These were phenotypically characterized, and whole-genome sequencing was performed. Mutations in promising targets or orthologue genes were inspected in Mtb clinical strains to establish potential associations between altered susceptibility to beta-lactams and the presence of key genomic signatures. The obtained isolates had substantial increases in the minimum inhibitory concentration of the selection antibiotic, and beta-lactam cross-resistance was detected in Mtb. Mutations in L,D-transpeptidases and major PBPs, canonical targets, or BlaC were not found. The transcriptional regulator PhoP (Rv0757) emerged as a common denominator for Mtb resistance to both amoxicillin and meropenem, while Rv2864c, a lipoprotein with PBP activity, appears to be specifically involved in decreased susceptibility to the carbapenem. Nonetheless, the mutational pattern detected in meropenem-resistant mutants was different from the yielded by amoxicillin-or vancomycin-selected isolates, suggesting that distinct pathways may participate in increased resistance to peptidoglycan inhibitors, including at the level of beta-lactam subclasses. Cross-resistance between beta-lactams and antimycobacterials was mostly unnoticed, and Msm meropenem-resistant mutants from parental strains with previous resistance to isoniazid or ethambutol were isolated at a lower frequency. Although cell-associated nitrocefin hydrolysis was increased in some of the isolates, our findings suggest that traditional assumptions of Mtb resistance relying largely in beta-lactamase activity and impaired access of hydrophilic molecules through lipid-rich outer layers should be challenged. Moreover, the therapeutical potential of the identified Mtb targets should be explored.This work was supported by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Switzerland, through Research Grant 2018, and by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through research project PTDC/BIA-MIC/31233/2017, awarded to MJC. FO (SFRH/BD/136853/2018) and CS (2021.05446.BD) are recipients of PhD fellowships from FCT.info:eu-repo/semantics/publishedVersio

Uncovering Beta-Lactam Susceptibility Patterns in Clinical Isolates of Mycobacterium tuberculosis through Whole-Genome Sequencing

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9431576/The increasing threat of drug resistance and a stagnated pipeline of novel therapeutics endanger the eradication of tuberculosis. Beta-lactams constitute promising additions to the current therapeutic arsenal and two carbapenems are included in group C of medicines recommended by the WHO for use in longer multidrug-resistant tuberculosis regimens. However, the determinants underlining diverse Mycobacterium tuberculosis phenotypes to beta-lactams remain largely undefined. To decipher these, we present a proof-of-concept study based on a large-scale beta-lactam susceptibility screening for 172 M. tuberculosis clinical isolates from Portugal, including 72 antimycobacterial drug-resistant strains. MICs were determined for multiple beta-lactams and strains were subjected to whole-genome sequencing to identify core-genome single-nucleotide variant-based profiles. Global and cell wall-targeted approaches were then followed to detect putative drivers of beta-lactam response. We found that drug-resistant strains were more susceptible to beta-lactams, but significant differences were not observed between distinct drug-resistance profiles. Sublineage 4.3.4.2 strains were significantly more susceptible to beta-lactams, while the contrary was observed for Beijing and 4.1.2.1 sublineages. While mutations in beta-lactamase or cell wall biosynthesis genes were uncommon, a rise in beta-lactam MICs was detected in parallel with the accumulation of mutations in peptidoglycan cross-linking or cell division genes. Finally, we exposed that putative beta-lactam resistance markers occurred in genes for which relevant roles in cell wall processes have been ascribed, such as rpfC or pknA. Genetic studies to validate the relevance of the identified mutations for beta-lactam susceptibility and further improvement of the phenotype-genotype associations are needed in the future. IMPORTANCE Associations between differential M. tuberculosis beta-lactam phenotypes and preexisting antimycobacterial drug resistance, strain sublineage, or specific mutational patterns were established. Importantly, we reveal that highly drug-resistant isolates of sublineage 4.3.4.2 have an increased susceptibility to beta-lactams compared with other strains. Thus, directing beta-lactams to treat infections by specific M. tuberculosis strains and refraining its use from others emerges as a potentially important strategy to avoid resistance development. Individual mutations in blaC or genes encoding canonical beta-lactam targets, such as peptidoglycan transpeptidases, are infrequent and do not greatly impact the MICs of potent carbapenem plus clavulanic acid combinations. An improved understanding of the global effect of cumulative mutations in relevant gene sets for peptidoglycan and cell division processes on beta-lactam susceptibility is also provided.This work was supported by Fundação para a Ciência e Tecnologia (PTDC/BIA-MIC/31233/2017 to M.J.C, SFRH/BD/136853/2018 to F.O. and 2021.05446.BD to C.S.) and by the European Society of Clinical Microbiology and Infectious Diseases (Research Grant 2018 to M.J.C.).info:eu-repo/semantics/publishedVersio

WGS-based dual strategy for the identification of key targets to enhance beta-lactam activity in mycobacterium tuberculosis

info:eu-repo/semantics/publishedVersio

Role of the N-glycolylation of mycobacterial peptidoglycan in host immune recognition and antibiotic resistance

Tese de mestrado, Ciências Biofarmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.Nos últimos anos, a tuberculose (TB) tornou-se uma emergência de saúde global, principalmente devido ao surgimento de estirpes multirresistentes e extensivamente resistentes do seu agente etiológico, Mycobacterium tuberculosis (Mtb). Este problema é acentuado pela relativa ineficácia da vacina BCG e dos agentes antimicobacterianos atualmente conhecidos e, pela falta de alternativas terapêuticas para a TB. Com quase dois milhões de mortes por TB anualmente, é urgente encontrar uma vacina eficaz contra a doença, desenvolver ferramentas de diagnóstico rápido e criar melhores esquemas de tratamento. Assim, é fundamental um melhor esclarecimento do mecanismo da patogénese da TB, que é atualmente mal compreendido, devido à estrutura altamente complexa da parede celular micobacteriana. A camada de peptidoglicano (PG) da parede celular micobacteriana é particularmente relevante, uma vez que apresenta modificações únicas, das quais a N-glicolilação do ácido murâmico, catalisada pela atividade da hidroxilase do ácido N-acetil murâmico (NamH), é de especial interesse. Estudos anteriores demonstraram que a N-glicolilação do PG promove a resistência a antibióticos β-lactâmicos e à lisozima. Da mesma forma, outros estudos demonstraram que a N-glicolilação do PG parece estimular a imunogenicidade da parede celular micobacteriana. Apesar de não ser um gene essencial, o namH é altamente conservado em estirpes clínicas de Mtb, o que indica que a N-glicolilação do PG possui uma função importante na resistência a antibióticos e/ou na patogénese da TB. Assim, o objetivo principal desta tese foi modular a expressão do namH de modo a compreender a importância da N-glicolilação do PG micobacteriano na resistência aos antibióticos e no reconhecimento das micobactérias por parte do hospedeiro. Para isso, vários mutantes knockdown do namH (namH-) foram construídos em M. smegmatis e M. tuberculosis H37Ra, usando uma nova e eficaz ferramenta molecular de regulação da transcrição, o CRISPR de interferência (CRISPRi). Esta técnica baseia-se na expressão induzida, com anidrotetraciclina (ATc), de um complexo dCas9-sgRNA para impedir a transcrição da sequência alvo, o que permite a investigação da função do gene de interesse, neste caso do namH. Os mutantes knockdown do namH construídos em M. smegmatis foram caracterizados através da realização de várias experiências: curvas de crescimento e plaqueamento de diluições seriadas de cada cultura numa área circular delimitada designada por spot. Estas experiências mostraram que quer a deleção quer a repressão do gene namH não origina defeitos graves no crescimento de M. smegmatis, confirmando-se assim que este gene não é essencial. Além disso, foi possível observar um nível residual de toxicidade da dCas9Sth1 durante as curvas de crescimento, ao verificar-se que o controlo M. smegmatis:PLJR962 cresce ligeiramente mais devagar na presença de indutor. Embora não tenha sido possível confirmar a repressão do namH em todos os mutantes knockdown, a repressão deste gene no mutante knockdown induzido M. smegmatis C2 ATc foi avaliada por PCR quantitativo em tempo real (qRT-PCR) e confirmada. Além disso, constatou-se que o CRISPRi provoca polaridade, ou seja, uma diminuição significativa da transcrição do gene a jusante. No entanto, não foi possível investigar de que modo a força da PAM escolhida, a cadeia de DNA alvo e o local alvo afetam a eficiência do CRISPRi. De seguida, o papel da N-glicolilação do PG na suscetibilidade a vários antibióticos foi investigado através da realização de ensaios para determinar a concentração mínima inibitória (CMI) e a concentração mínima bactericida (CMB) de diversos β-lactâmicos (amoxicilina, cefotaxima, meropenem), na presença e ausência do inibidor de β-lactamase clavulanato, e de alguns agentes antimicobacterianos (etambutol e isoniazida) para todos os mutantes knockdown em M. smegmatis, na presença e ausência de indutor. Os resultados obtidos mostraram que o meropenem é mais eficiente ao inibir o crescimento das micobactérias do que a amoxicilina ou a cefotaxima. Este efeito parece ser causado pela resistência inerente do meropenem às β-lactamases micobacterianas e, também, devido a uma inibição simultânea de L,D-transpeptidases e, algumas D,D-transpeptidases e D,D-carboxipeptidases. Além disso, constatou-se que a N-glicolilação do PG tem um papel significativo na resistência aos β-lactâmicos, uma vez que tanto o mutante de deleção do namH (ΔnamH) quanto os mutantes knockdown induzidos exibiram um fenótipo de suscetibilidade à maioria dos β-lactâmicos. Da mesma forma, foi observada uma correlação relevante entre a N-glicolilação do PG e o mecanismo de ação da cefotaxima pois os mutantes namH- exibiram uma diminuição particularmente significativa da CMI da cefotaxima ou da cefotaxima-clavulanato na presença de indutor. Verificou-se ainda que a principal β-lactamase de M. smegmatis (BlaS) hidrolisa os antibióticos β-lactâmicos com diferente eficiência, pois a adição de clavulanato aos β-lactâmicos diminuiu consideravelmente a CMI da amoxicilina, o que não aconteceu no caso da cefotaxima e do meropenem. Os ensaios de CMB mostraram que todos os β-lactâmicos possuem atividade bactericida para a maioria das amostras de M. smegmatis testadas, o que indica que a N-glicolilação do PG não afeta consideravelmente a capacidade dos β-lactâmicos matarem as micobactérias. No entanto, verificou-se que a indução do sistema de CRISPRi pareceu aumentar a atividade bactericida dos β-lactâmicos em alguns casos, em que os mutantes knockdown do namH eram mais facilmente mortos na presença de indutor. Os ensaios de difusão em disco permitiram a determinação das zonas de inibição do meropenem para todos os mutantes knockdown construídos em M. smegmatis, com e sem indutor. Tal como tinha sido demonstrado anteriormente, os resultados destes testes sugeriram que a proteína NamH desempenha um papel na resistência ao meropenem, pois tanto o mutante de deleção do namH (ΔnamH) quanto os mutantes de knockdown do namH induzidos exibiram um fenótipo exacerbado de hipersuscetibilidade ao meropenem. Estas experiências demonstraram que os mutantes de deleção/repressão do namH são mais suscetíveis à maioria dos β-lactâmicos. Este “fenótipo de hipersuscetibilidade aos β-lactâmicos” pode, então, ser explicado pela inibição: i) da atividade hidrolítica de β-lactamase da NamH; ii) da atividade de monooxigenase/hidroxilase da NamH, que modifica os açúcares do PG micobacteriano; iii) ambas as atividades, permitindo uma sinergia entre a inibição da atividade de β-lactamase e a inibição da N-glicolilação do PG. De facto, a redução da atividade de hidroxilase da NamH tem como consequências: i) a diminuição da integridade do PG devido a uma menor disponibilidade de grupos N-glicolil para estabelecer ligações de hidrogénio; ii) a alteração da polimerização do PG devido a uma menor disponibilidade de precursores do PG que estejam N-glicolilados. Assim, a deleção/repressão do namH pode ser utilizada em sinergia com o tratamento com β-lactâmicos para diminuir a integridade do PG micobacteriano. A contribuição da N-glicolilação do PG para o reconhecimento pelo sistema imunitário do hospedeiro foi avaliada através da realização de infeções de macrófagos de ratinho RAW 264.7 com um mutante namH- selecionado, M. smegmatis C2. Verificou-se que os macrófagos são mais eficazes a erradicar o mutante de knockdown induzido C2 ATc do que qualquer outra bactéria no t=24h, o que sugere que a presença do PG N-glicolilado promove a sobrevivência intracelular, possivelmente ao aumentar o fitness das micobactérias dentro dos macrófagos. Esta observação parece indicar que a N-glicolilação do PG provoca um menor reconhecimento das micobactérias por parte dos macrófagos, o que não é unânime visto que grande parte dos estudos anteriores verificou que a N-glicolilação do PG potencia o reconhecimento imunológico e a subsequente resposta imune. Em conclusão, o trabalho apresentado nesta tese ajudou a esclarecer o papel da N-glicolilação do PG na suscetibilidade aos antibióticos, ao levantar novas hipóteses sobre a forma como a proteína NamH promove a resistência aos β-lactâmicos. Do mesmo modo, esta tese também levantou questões importantes sobre o papel da N-glicolilação do PG nas interações patogénio-hospedeiro, nomeadamente ao nível do reconhecimento imune. Os resultados apresentados e as conclusões alcançadas poderão ser relevantes para uma melhor compreensão da patogénese da TB e para o desenvolvimento de terapêuticas alternativas para o tratamento da TB, no futuro.n the past years, tuberculosis (TB) has become a global health emergency, namely due to the emergence of multi-drug and extensively-drug resistant strains of its etiologic agent, Mycobacterium tuberculosis (Mtb). This problem is accentuated by the relative ineffectiveness of the BCG vaccine and of currently known antimycobacterial agents and, by the lack of alternative TB therapeutics. The pathogenesis of Mtb is poorly understood due to the highly complex structure of the cell wall (CW). The peptidoglycan (PG) layer of the mycobacterial CW is specifically relevant since it features unique modifications, of which the N-glycolylation of PG muramic acid, catalyzed by the activity of the N-acetyl muramic acid hydroxylase (NamH), is of special interest. Previous studies have demonstrated that the N-glycolylation of PG increases β-lactams/lysozyme resistance as well as the immunogenicity of the mycobacterial CW. Despite not being essential, the namH gene is highly conserved in Mtb clinical isolates, implying a vital function for the N-glycolylation of PG in antibiotic resistance or/and in TB pathogenesis. Therefore, the main goal of this thesis was to modulate namH expression in order to understand the significance of the N-glycolylation of mycobacterial PG in antibiotic resistance and in host immune recognition. To do that, several namH knockdown (namH-) mutants were constructed in both M. smegmatis and M. tuberculosis H37Ra, using a new effective transcription regulation tool, CRISPR interference (CRISPRi). This technique employs a dCas9-sgRNA complex to repress any target gene by sterically hindering its transcription, thus, enabling the inquiry of the functional significance of namH. The M. smegmatis namH- mutants were phenotypically characterized by performing growth curves and spotting dilutions. These experiments demonstrated that namH knockout/repression does not produce severe growth defects, thus, confirming the non-essentiality of namH. Moreover, a residual level of dCas9Sth1 toxicity was uncovered by performing growth curves. Although it was not possible to confirm the repression of namH in all knockdown mutants, the repression of namH in the induced knockdown mutant M. smegmatis C2 in the presence of anhydrotetracycline (ATc) was assessed by quantitative real-time PCR (qRT-PCR) and confirmed. Furthermore, CRISPRi was found to cause a significant level of downstream polarity. Nevertheless, it was not possible to probe how the strength of the employed PAM, the targeted DNA strand and the target site affect the efficiency of CRISPRi. In addition, the role of the N-glycolylation of PG in antibiotic susceptibility was determined by performing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays for all M. smegmatis namH knockdown mutants, with and without inducer (ATc). The MIC assays showed that meropenem is more efficient at inhibiting the growth of mycobacteria, when compared to amoxicillin or cefotaxime. Besides, the N-glycolylation of PG was found to have a role in β-lactams resistance, as both M. smegmatis ΔnamH and the induced knockdown mutants exhibited increased susceptibility to most β-lactams. Likewise, a strong link between the N-glycolylation of PG and the mechanism of action of cefotaxime was uncovered, as the namH knockdown mutants displayed a particularly significant decrease in MIC values for cefotaxime or cefotaxime-clavulanate in the presence of inducer. Furthermore, differences in the hydrolytic efficiency of the main β-lactamase of M. smegmatis (BlaS) to various β-lactams were verified as the addition of clavulanate to β-lactams considerably decreased the MIC of amoxicillin, as opposed to cefotaxime and meropenem. The MBC assays showed that all β-lactams were bactericidal against most of the tested M. smegmatis samples, indicating that the N-glycolylation of PG does not severely affect the killing activity of β-lactams. Subsequently, the disk diffusion assays enabled the assessment of the meropenem zones of inhibition for all M. smegmatis knockdown mutants, with and without inducer. These experiments suggested that NamH plays a role in meropenem resistance as both M. smegmatis ΔnamH and the induced namH knockdown mutants displayed an exacerbated meropenem hypersusceptibility phenotype. Overall, these antibiotic susceptibility tests demonstrated that the namH knockout/knockdown mutants display increased susceptibility to most β-lactams. This “β-lactams hypersusceptibility phenotype” may be explained by the inhibition of: i) the hydrolytic activity of NamH as a β-lactamase; ii) the activity of NamH as a monooxygenase, which modifies PG sugars; iii) both activities, enabling a synergy between the inhibition of β-lactamase activity and the inhibition of the N-glycolylation of PG. Indeed, the reduced hydroxylase activity of NamH can lead to: i) decreased PG integrity due to a lower availability of N-glycolyl groups to establish hydrogen bonds; ii) altered PG polymerization due to a lower availability of N-glycolylated PG precursors. The contribution of the N-glycolylation of PG in host immune recognition and pathogenesis was assessed by performing infections of RAW 264.7 macrophages with a selected namH- mutant. Macrophages were found to be more effective at killing the induced knockdown mutant C2 ATc than any other bacteria at t=24h, suggesting that the presence of N-glycolylated PG promotes intracellular survival, possibly by increasing the fitness of mycobacteria inside macrophages. In conclusion, the work presented in this thesis helped enlighten the role of the N-glycolylation of PG in antibiotic susceptibility by posing new hypothesis as to how NamH enables β-lactams resistance and raised important questions about the role of the N-glycolylation of PG in host-pathogen interactions, which may lead to a better understanding of TB pathogenesis in the future.Com o patrocínio da Faculdade de Farmácia da Universidade de Lisboa e do iMed.ULisboa

CRISPRi-mediated characterization of novel anti-tuberculosis targets: Mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survival

The lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications