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The emerging fungal pathogen Candida auris is known for its strong skin tropism and resilience against antifungal and disinfection treatment, posing a significant challenge for healthcare units. Although efforts to identify the effectors of its unique pathogenic behavior have been insightful, the role of the high-osmolarity glycerol (HOG) pathway in this context remains unexplored. The study by Shivarathri and co-workers (R. Shivarathri, M. Chauhan, A. Datta, D. Das et al., mBio 15:e02748-24, 2024, https://doi.org/10.1128/mbio.02748-24) sought to address this gap. This report indeed advances our understanding of the critical role of the HOG pathway in C. auris pathogenicity by emphasizing its involvement in skin colonization, biofilm formation, and evasion of phagocyte attack.

Mutations affecting Clostridioides difficile flagellin (FliC) have been shown to be hypervirulent in animal models and display increased toxin production and alterations in central metabolism. The regulation of flagellin levels in bacteria is governed by a tripartite regulatory network involving fliC, fliW, and csrA, which creates a feedback system to regulate flagella production. Through genomic analysis of C. difficile clade 5 strains (non-motile), we identified they have jettisoned many of the genes required for flagellum biosynthesis yet retain the major flagellin gene fliC and regulatory gene fliW. We therefore investigated the roles of fliC, fliW, and csrA in the clade 5 ribotype 078 strain C. difficile 1015, which lacks flagella and is non-motile. Analysis of mutations in fliC, fliW, and csrA (and all combinations) on C. difficile pathogenesis indicated that FliW plays a central role in C. difficile virulence as animals infected with strains carrying a deletion of fliW showed decreased survival and increased disease severity. These in vivo findings were supported by in vitro studies showing that mutations impacting the activity of FliW showed increased toxin production. We further identified that FliW can interact with the toxin-positive regulator TcdR, indicating that modulation of toxin production via FliW occurs by sequestering TcdR from activating toxin transcription. Furthermore, disruption of the fliC-fliW-csrA network results in significant changes in carbon source utilization and sporulation. This work highlights that key proteins involved in flagellar biosynthesis retain their regulatory roles in C. difficile pathogenesis and physiology independent of their functions in motility.

Importance: Clostridioides difficile is a leading cause of nosocomial antibiotic-associated diarrhea in developed countries with many known virulence factors. In several pathogens, motility and virulence are intimately linked by regulatory networks that allow coordination of these processes in pathogenesis and physiology. Regulation of C. difficile toxin production by FliC has been demonstrated in vitro and in vivo and has been proposed to link motility and virulence. Here, we show that clinically important, non-motile C. difficile strains have conserved FliC and regulatory partners FliW and CsrA, despite lacking the rest of the machinery to produce functional flagella. Our work highlights a novel role for flagellin outside of its role in motility and FliW in the pathogenesis and physiology of C. difficile.

Microbial impact on tumorigenesis of heritable cancers proximal to the gut is well-documented. Whether the microbiota influences cancers arising from inborn mutations at sites distal to the gut is undetermined. Using two models of heritable cancer, Trp53-deficient mice and Wnt1-transgenic mice, and a gnotobiotic approach, we found the microbiota to be inconsequential for tumor development. This work furthers our understanding of the degree of the microbial impact on tumor development.

Importance: The influence of the microbiome on the development of cancer is well-documented with many if not all published studies reporting either a positive or a negative impact. None of the published studies, however, presented data on the influence of the microbiome on the development of heritable cancer. We find that the microbiota has no influence on cancer development in two models of spontaneous cancers driven by germline Trp53 deficiency and constitutive Wnt1 signaling.

Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet ("β barrel") that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called the barrel assembly machine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of the Escherichia coli OmpC protein into the OM in vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called the barrel assembly machine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues in Escherichia coli OmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane.

Streptolysin O (SLO) is a virulence determinant of group A Streptococcus (S. pyogenes), the agent of streptococcal sore throat and severe invasive infections. SLO is a member of a family of bacterial pore-forming toxins known as cholesterol-dependent cytolysins, which require cell membrane cholesterol for pore formation. While cholesterol is essential for cytolytic activity, accumulating data suggest that cell surface glycans may also participate in the binding of SLO and other cholesterol-dependent cytolysins to host cells. Here, we find that unbiased CRISPR screens for host susceptibility factors for SLO cytotoxicity identified genes encoding enzymes involved in the earliest steps of glycosphingolipid (GSL) biosynthesis. Targeted knockouts of these genes conferred relative resistance to SLO cytotoxicity in two independent human cell lines. Inactivation of ugcg, which codes for UDP-glucose ceramide glucosyltransferase, the enzyme catalyzing the first glycosylation step in GSL biosynthesis, reduced the clustering of SLO on the cell surface. This result suggests that binding to GSLs serves to cluster SLO molecules at lipid rafts where both GSLs and cholesterol are abundant. SLO clustering and susceptibility to SLO cytotoxicity were restored by reconstituting the GSL content of ugcg knockout cells with ganglioside GM1, but susceptibility to SLO cytotoxicity was not restored by a GM1 variant that lacks an oligosaccharide head group required for SLO binding, nor by a variant with a "kinked" acyl chain that prevents efficient packing of the ganglioside ceramide moiety with cholesterol. Thus, SLO appears to co-opt cell surface glycosphingolipids to gain access to lipid rafts for increased efficiency of pore formation and cytotoxicity.

Importance: Group A Streptococcus is a global public health concern as it causes streptococcal sore throat and less common but potentially life-threatening invasive infections. Invasive infections have been associated with bacterial strains that produce large amounts of a secreted toxin, streptolysin O (SLO), which belongs to a family of pore-forming toxins produced by a variety of bacterial species. This study reveals that SLO binds to a class of molecules known as glycosphingolipids on the surface of human cells and that this interaction promotes efficient binding of SLO to cholesterol in the cell membrane and enhances pore formation. Understanding how SLO damages human cells provides new insight into streptococcal infection and may inform new approaches to treatment and prevention.

Peptidoglycan (PG) is an important bacterial macromolecule that confers cell shape and structural integrity, and is a key antibiotic target. Its synthesis and turnover are carefully coordinated with other cellular processes and pathways. Despite established connections between the biosynthesis of PG and the outer membrane, or PG and DNA replication, links between PG and folate metabolism remain comparatively unexplored. Folate is an essential cofactor for bacterial growth and is required for the synthesis of many important metabolites. Here we show that inhibition of folate synthesis in the important Gram-negative pathogen Pseudomonas aeruginosa has downstream effects on PG metabolism and integrity that can manifest as the formation of a subpopulation of round cells that can undergo explosive lysis. Folate inhibitors potentiated β-lactams by perturbation of PG recycling, reducing expression of the AmpC β-lactamase. Supporting this mechanism, folate inhibitors also synergized with fosfomycin, an inhibitor of MurA, the first committed step in PG synthesis that can be bypassed by PG recycling. These insights led to the design of a dual-active inhibitor that overcomes NDM-1 metallo-β lactamase-mediated meropenem resistance and synergizes with the folate inhibitor, trimethoprim. We show that folate and PG metabolism are intimately connected, and targeting this connection can overcome antibiotic resistance in Gram-negative pathogens.

Importance: To combat the alarming global increase in superbugs amid the simultaneous scarcity of new drugs, we can create synergistic combinations of currently available antibiotics or chimeric molecules with dual activities, to minimize resistance. Here we show that older anti-folate drugs synergize with specific cell wall biosynthesis inhibitors to kill the priority pathogen, Pseudomonas aeruginosa. Anti-folate drugs caused a dose-dependent loss of rod cell shape followed by explosive lysis, and synergized with β-lactams that target D,D-carboxypeptidases required to tailor the cell wall. Anti-folates impaired cell wall recycling and subsequent downstream expression of the chromosomally encoded β-lactamase, AmpC, which normally destroys β-lactam antibiotics. Building on the anti-folate-like scaffold of a metallo-β-lactamase inhibitor, we created a new molecule, MLLB-2201, that potentiates β-lactams and anti-folates and restores meropenem activity against metallo-β-lactamase-expressing Escherichia coli. These strategies are useful ways to tackle the ongoing rise in dangerous bacterial pathogens.

Sepsis-induced acute liver injury (SALI) is a prevalent and life-threatening complication associated with sepsis. The gut microbiota plays a crucial role in the maintenance of health and the development of diseases. The impact of physical exercise on gut microbiota modulation has been well-documented. However, the potential impact of gut microbiome on exercise training-induced protection against SALI remains uncertain. Here, we discovered exercise training ameliorated SALI and systemic inflammation in septic mice. Notably, gut microbiota pre-depletion abolished the protective effects of exercise training in SALI mice. Fecal microbiota transplantation treatment revealed that exercise training-associated gut microbiota contributed to the beneficial effect of exercise training on SALI. Exercise training modulated the metabolism of Ligilactobacillus and enriched betulinic acid (BA) levels in mice. Functionally, BA treatment conferred protection against SALI by inhibiting the hepatic inflammatory response in mice. BA bound and inactivated hnRNPA2B1, thus suppressing NLRP3 inflammasome activation in macrophages. Collectively, this study reveals gut microbiota is involved in the protective effects of exercise training against SALI, and gut microbiota-derived BA inhibits the hepatic inflammatory response via the hnRNPA2B1-NLRP3 axis, providing a potential therapeutic strategy for SALI.

Importance: Sepsis is characterized by a dysregulated immune response to an infection that leads to multiple organ dysfunction. The occurrence of acute liver injury is frequently observed during the initial stage of sepsis and is directly linked to mortality in the intensive care unit. The preventive effect of physical exercise on SALI is well recognized, yet the underlying mechanism remains poorly elucidated. Exercise training alters the gut microbiome in mice, increasing the abundance of Ligilactobacillus and promoting the generation of BA. Additionally, BA supplementation can suppress the NLRP3 inflammasome activation in macrophages by directly binding to hnRNPA2B1, thereby mitigating SALI. These results highlight the beneficial role of gut microbiota-derived BA in inhibiting the hepatic inflammatory response, which represents a crucial stride toward implementing microbiome-based therapeutic strategies for the clinical management of sepsis.

Deoxynivalenol (DON), a mycotoxin primarily produced by Fusarium species, is commonly found in cereal grains and poses risks to human and animal health, as well as global grain trade. This study demonstrates that methyl jasmonate (MeJA), a natural plant hormone, inhibits the growth and conidiation of Fusarium graminearum. Importantly, MeJA significantly reduces DON production by suppressing TRI gene expression and toxisome formation. To explore the molecular mechanism, we identified MeJA-tolerant mutants, including a transcription factor MRT1 and cAMP-PKA pathway-related genes (FgGPA1 and FgSNT1). MeJA treatment reduced PKA activity and intracellular cAMP levels in F. graminearum, suggesting it targets the cAMP-PKA pathway. Notably, the MeJA-resistant mutant FgGPA1^R178H enhanced fungal growth, DON production, and cAMP levels in the presence of MeJA. Exogenous cAMP alleviated MeJA's inhibitory effects on DON production, further supporting this pathway's involvement. Interestingly, MeJA had no effect on all three MAP kinase pathways (Mgv1, Gpmk1, and FgHog1). Truncated and phospho-mimicking mutations in Mrt1 or FgSnt1 conferred MeJA resistance, suggesting they may act downstream of the cAMP-PKA pathway. In conclusion, MeJA presents a promising approach to control F. graminearum growth and DON production.IMPORTANCEDeoxynivalenol (DON) poses significant risks to both human and animal health and severely disrupts the global grain trade due to its prevalence as a common contaminant in wheat grains. With rising public concern over food safety, finding effective and sustainable methods to reduce DON contamination becomes increasingly urgent. In our study, we found that methyl jasmonate (MeJA), a natural plant hormone, can effectively inhibit the vegetative growth of F. graminearum and significantly reduce its DON toxin production. To explore the underlying molecular mechanism, we identified the mutations in MeJA-tolerant mutants and revealed that MeJA effectively exerts its antifungal activities by inhibiting the cAMP-PKA signaling pathway in F. graminearum. Our work provides a promising natural solution to reduce DON toxin contamination in cereal grains, enhancing food safety while decreasing the reliance on chemical fungicides and their associated environmental impact.

A quarter of a century ago, Liise-anne Pirofski and Arturo Casadevall shared their concepts of microbial pathogenesis through the lens of a damage-response framework (DRF), which characterizes disease by assessing the dynamic interactions between the host and pathogen as reflected by damage as the readout. This framework has evolved to be a powerful tool for understanding the biology of complex infectious diseases, analyzing emerging and reemerging microbes, and developing therapeutic approaches to combat infections. The DRF is also frequently used to explain research at scientific meetings and to teach microbial pathogenesis to diverse learners. This mGem reviews how the DRF came to be and provides an overview of how it is used. Without a doubt, the scientific community will continue to leverage the DRF to advance research and innovate therapeutic approaches, which is especially important as new and reemerging infectious diseases threaten global health.

Anaplasma phagocytophilum is an obligate intracellular rickettsial pathogen that infects humans and animals. The black-legged tick Ixodes scapularis acts as a vector and transmits this bacterium to the vertebrate host. Upon entry into a host cell, A. phagocytophilum resides and multiplies in a host-derived vacuole called morulae. There is not much information available on the molecules that play an important role(s) in A. phagocytophilum entry and formation of these morulae in tick cells. In this study, we provide evidence that tick vesicular-associated membrane proteins, VAMP3 and VAMP4, play important roles in this phenomenon. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis showed that both vamp3 and vamp4 transcripts are significantly upregulated at early time points of A. phagocytophilum infection in tick cells. We noted that both VAMP3 and VAMP4 predominantly localized to the A. phagocytophilum-containing vacuole. RNAi-mediated silencing of vamp3 and/or vamp4 expression, followed by confocal microscopy and expression analysis, indicated an impairment in A. phagocytophilum morulae formation in tick cells. We also noted that VAMP3 and VAMP4 play a role in the A. phagocytophilum persistent infection of ticks and tick cells. Furthermore, RNAi-mediated silencing of expression of arthropod vamp3 and vamp4 affected bacterial acquisition from an infected murine host to ticks. Collectively, this study not only provides evidence on the role of arthropod vesicular-associated membrane proteins in A. phagocytophilum morulae formation in tick cells but also demonstrates that these proteins are important for bacterial acquisition from an infected vertebrate host into ticks.

Importance: Anaplasma phagocytophilum is a tick-borne pathogen primarily transmitted by black-legged Ixodes scapularis ticks to humans and animals. This bacterium enters host cells, forms a host-derived vacuole, and multiplies within this vacuole. The molecules that are critical in the formation of host-derived vacuole in tick cells is currently not well-characterized. In this study, we provide evidence that arthropod vesicular-associated membrane proteins, VAMP3 and VAMP4, are critical for A. phagocytophilum early and persistent infection in tick cells. These arthropod proteins are important for the formation of host-derived vacuoles in tick cells. Our study also provides evidence that these proteins are important for A. phagocytophilum acquisition from the infected murine host into ticks. Characterization of tick molecules important in bacterial entry and/or survival in the vector host could lead to the development of strategies to target this and perhaps other rickettsial pathogens.