Genetic variation of DNA methyltransferase-3A contributes to protection against persistent MRSA bacteremia in patients.

The role of the host in development of persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is not well understood. A cohort of prospectively enrolled patients with persistent methicillin-resistant S. aureus bacteremia (PB) and resolving methicillin-resistant S. aureus bacteremia (RB) matched by sex, age, race, hemodialysis status, diabetes mellitus, and presence of implantable medical device was studied to gain insights into this question. One heterozygous g.25498283A > C polymorphism located in the DNMT3A intronic region of chromosome 2p with no impact in messenger RNA (mRNA) expression was more common in RB (21 of 34, 61.8%) than PB (3 of 34, 8.8%) patients (P = 7.8 × 10-6). Patients with MRSA bacteremia and g.25498283A > C genotype exhibited significantly higher levels of methylation in gene-regulatory CpG island regions (Δmethylation = 4.1%, P < 0.0001) and significantly lower serum levels of interleukin-10 (IL-10) than patients with MRSA bacteremia without DNMT3A mutation (A/C: 9.7038 pg/mL vs. A/A: 52.9898 pg/mL; P = 0.0042). Expression of DNMT3A was significantly suppressed in patients with S. aureus bacteremia and in S. aureus-challenged primary human macrophages. Small interfering RNA (siRNA) silencing of DNMT3A expression in human macrophages caused increased IL-10 response upon S. aureus stimulation. Treating macrophages with methylation inhibitor 5-Aza-2'-deoxycytidine resulted in increased levels of IL-10 when challenged with S. aureus In the murine sepsis model, methylation inhibition increased susceptibility to S. aureus These findings indicate that g.25498283A > C genotype within DNMT3A contributes to increased capacity to resolve MRSA bacteremia, potentially through a mechanism involving increased methylation of gene-regulatory regions and reduced levels of antiinflammatory cytokine IL-10.

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism.

Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence.

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

Coupling capillary zone electrophoresis with electron transfer dissociation and activated ion electron transfer dissociation for top-down proteomics.

Top-down proteomics offers the potential for full protein characterization, but many challenges remain for this approach, including efficient protein separations and effective fragmentation of intact proteins. Capillary zone electrophoresis (CZE) has shown great potential for separation of intact proteins, especially for differentially modified proteoforms of the same gene product. To date, however, CZE has been used only with collision-based fragmentation methods. Here we report the first implementation of electron transfer dissociation (ETD) with online CZE separations for top-down proteomics, analyzing a mixture of four standard proteins and a complex protein mixture from the Mycobacterium marinum bacterial secretome. Using a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate that CZE is fully compatible with ETD as well as higher energy collisional dissociation (HCD), and that the two complementary fragmentation methods can be used in tandem on the electrophoretic time scale for improved protein characterization. Furthermore, we show that activated ion electron transfer dissociation (AI-ETD), a recently introduced method for enhanced ETD fragmentation, provides useful performance with CZE separations to greatly increase protein characterization. When combined with HCD, AI-ETD improved the protein sequence coverage by more than 200% for proteins from both standard and complex mixtures, highlighting the benefits electron-driven dissociation methods can add to CZE separations.

A novel ESX-1 locus reveals that surface-associated ESX-1 substrates mediate virulence in Mycobacterium marinum.

EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.

Homeostasis of N-α-terminal acetylation of EsxA correlates with virulence in Mycobacterium marinum.

The mycobacterial Esx-1 (ESAT-6 system 1) exporter translocates virulence factors across the cytoplasmic membrane to the cell wall, cell surface, and the bacteriological medium in vitro. The mechanisms underlying substrate targeting to distinct locations are unknown. Several Esx-1 substrates are N-α-terminally acetylated. The role of this rare modification in bacteria is unclear. We sought to identify genes required for Esx-1 substrate modification, transport, and localization. Pathogenic mycobacteria lyse Acanthamoeba castellanii in an Esx-1-dependent manner. We conducted a genetic screen to identify Mycobacterium marinum strains which failed to lyse amoebae. We identified a noncytotoxic M. marinum strain with a transposon insertion in a predicted N-α-terminal acetyltransferase not previously linked to mycobacterial pathogenesis. Disruption of this gene led to attenuation of virulence, failure to induce a type I interferon response during macrophage infection, and loss of hemolytic activity. The major Esx-1 substrates, EsxA and EsxB, were exported to the cell surface, but only low levels were released into the bacteriological medium. The balance of EsxA N-α-terminal acetylation was disrupted, resulting in a mycobacterial strain in which surface-associated EsxA was hyperacetylated. Genetic complementation completely restored Esx-1 function and the levels of N-α-terminally acetylated EsxA on the surface but restored only low levels of Esx-1 substrates in the bacteriological medium. Our results reveal a novel gene required for mycobacterial Esx-1 export. Our findings indicate that maintaining the homeostasis of Esx-1 substrate N-α-terminal acetylation is essential for Esx-1-mediated virulence. We propose an inverse correlation between EsxA acetylation and virulence.

Predicting Clinical Outcomes of SARS-CoV-2 Drug Efficacy with a High-Throughput Human Airway Microphysiological System.

The average cost to bring a new drug from its initial discovery to a patient's bedside is estimated to surpass $2 billion and requires over a decade of research and development. There is a need for new drug screening technologies that can parse drug candidates with increased likelihood of clinical utility early in development in order to increase the cost-effectiveness of this pipeline. For example, during the COVID-19 pandemic, resources were rapidly mobilized to identify effective therapeutic treatments but many lead antiviral compounds failed to demonstrate efficacy when progressed to human trials. To address the lack of predictive preclinical drug screening tools, PREDICT96-ALI, a high-throughput (n = 96) microphysiological system (MPS)  that recapitulates primary human tracheobronchial tissue,is adapted for the evaluation of differential antiviral efficacy of native SARS-CoV-2 variants of concern. Here, PREDICT96-ALI resolves both the differential viral kinetics between variants and the efficacy of antiviral compounds over a range of drug doses. PREDICT96-ALI is able to distinguish clinically efficacious antiviral therapies like remdesivir and nirmatrelvir from promising lead compounds that do not show clinical efficacy. Importantly, results from this proof-of-concept study track with known clinical outcomes, demonstrate the feasibility of this technology as a prognostic drug discovery tool.

A High-Throughput, High-Containment Human Primary Epithelial Airway Organ-on-Chip Platform for SARS-CoV-2 Therapeutic Screening.

COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack of human-relevant preclinical models compatible with therapeutic screening on the native virus, which requires a high-containment environment. Here, we report SARS-CoV-2 infection and robust viral replication in PREDICT96-ALI, a high-throughput, human primary cell-based organ-on-chip platform. We evaluate unique infection kinetic profiles across lung tissue from three human donors by immunofluorescence, RT-qPCR, and plaque assays over a 6-day infection period. Enabled by the 96 devices/plate throughput of PREDICT96-ALI, we also investigate the efficacy of Remdesivir and MPro61 in a proof-of-concept antiviral study. Both compounds exhibit an antiviral effect against SARS-CoV-2 in the platform. This demonstration of SARS-CoV-2 infection and antiviral dosing in a high-throughput organ-on-chip platform presents a critical capability for disease modeling and therapeutic screening applications in a human physiology-relevant in vitro system.

Mycobacterium tuberculosis Rv1090 and Rv1987 encode functional ß-glucan-targeting proteins.

Mycobacterium tuberculosis is a facultative intracellular pathogen, and the ability of this bacterium to survive and to grow inside macrophages is central to its virulence. Multiple strategies are employed by M. tuberculosis to ensure survival in macrophages, including secretion of several proteins, which are good candidates to be virulence factors, drug targets for disease intervention, and vaccine antigens. However, some M. tuberculosis secreted proteins do not appear to play any role in the growth or survival of the bacterium in its mammalian host. Among these proteins are three putative cellulose-targeting proteins encoded by the genes Rv0062, Rv1090, and Rv1987. It has been previously shown that Rv0062 encodes an active cellulase. Here we report that Rv1090 and Rv1987 also encode functional proteins. Rv1090 is able to hydrolyze barley ß-glucan while Rv1987 displays cellulose-binding activity on filter paper and on microcrystalline cellulose (Avicel). Collectively, these observations point toward a unique unknown relationship between M. tuberculosis and a cellulose-containing host. We hypothesize that amoeba could be such hosts.

Paradoxical conservation of a set of three cellulose-targeting genes in Mycobacterium tuberculosis complex organisms.

The genome of the tuberculosis agent Mycobacterium tuberculosis encodes a putative cellulose-binding protein (CBD2), one candidate cellulase (Cel12), and one fully active cellulase (Cel6). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. In order to investigate the biological role of such cellulose-targeting genes in M. tuberculosis we report here the search for and transcription analysis of this set of genes in the genus Mycobacterium. An in silico search for cellulose-targeting orthologues found that only 2.5 % of the sequenced bacterial genomes encode the Cel6, Cel12 and CBD2 gene set simultaneously, including those of the M. tuberculosis complex (MTC) members. PCR amplification and sequencing further demonstrated the presence of these three genes in five non-sequenced MTC bacteria. Among mycobacteria, the combination of Cel6, Cel12 and CBD2 was unique to MTC members, with the exception of Mycobacterium bovis BCG Pasteur, which lacked CBD2. RT-PCR in M. tuberculosis H37Rv indicated that the three cellulose-targeting genes were transcribed into mRNA. The present work shows that MTC organisms are the sole mycobacteria among very few organisms to encode the three cellulose-targeting genes CBD2, Cel6 and Cel12. Our data point toward a unique, yet unknown, relationship with non-plant cellulose-producing hosts such as amoebae.

Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus.

Mass spectrometry (MS) for the detection of proteins is an indispensable tool for evaluating the biological processes of the proteome. Proteomics frequently requires proteolysis of proteins into peptide fragments. Proteins can be refractory to ideal proteolysis at the sequence level rendering them difficult to analyze by routine proteomics methods. EsxA (ESAT-6, Early Secreted Antigen, 6kDa) is a major virulence determinant of Mycobacterium tuberculosis, the cause of human tuberculosis. EsxA is routinely used to evaluate mycobacterial virulence in the laboratory and as a biomarker for tuberculosis in humans. The sequence of EsxA hinders deeper MS analysis beyond routine detection. Here we engineer the sequence of EsxA to add desirable tryptic properties aimed at improving complex MS analysis. We demonstrate that EsxA variants are amenable to MS analysis and remain functional in established in vitro and ex vivo assays of Esx-1-function. We provide the first demonstration of molecular engineering to specifically improve MS analysis of individual microbial proteins.

Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis.

BACKGROUND: Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival. METHODOLOGY/PRINCIPAL FINDINGS: Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc(2) 155, M. smegmatis ATCC 19420(T) and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth. CONCLUSIONS/SIGNIFICANCE: Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.

Genome analyses highlight the different biological roles of cellulases.

Cellulolytic enzymes have been the subject of renewed interest owing to their potential role in the conversion of plant lignocellulose to sustainable biofuels. An analysis of ∼1,500 complete bacterial genomes, presented here, reveals that ∼40% of the genomes of sequenced bacteria encode at least one cellulase gene. Most of the bacteria that encode cellulases are soil and marine saprophytes, many of which encode a range of enzymes for cellulose hydrolysis and also for the breakdown of the other constituents of plant cell walls (hemicelluloses and pectins). Intriguingly, cellulases are present in organisms that are usually considered as non-saprophytic, such as Mycobacterium tuberculosis, Legionella pneumophila, Yersinia pestis and even Escherichia coli. We also discuss newly emerging roles of cellulases in such non-saprophytic organisms.

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms.

BACKGROUND: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii. METHODOLOGY/PRINCIPAL FINDINGS: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6)) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10(4) CFU/mL) than M. tuberculosis (42×10(1) CFU/mL) and M. bovis (35×10(1) CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde. CONCLUSIONS/SIGNIFICANCE: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.