Journal of Bacteriology最新文献

Uropathogenic Escherichia coli (UPEC) strains that express the K1 capsule are associated with severe invasive disease, including pyelonephritis, urosepsis, and neonatal meningitis. The K1 capsule can be modified by NeuO, a phage-encoded phase-variable O-acetyltransferase. The role of O-acetylation in pathogenesis of K1 UPEC remains to be fully elucidated. Here, we assessed the prevalence of the neuO gene in a K1 E. coli data set comprising 8,659 genomes and observed that 43.5% of genomes harbor neuO, with a high prevalence (88.1%) in the pandemic UPEC clone sequence type (ST) 95. We generated an isogenic ∆neuO mutant in the reference ST95 strain MS7163 and complemented this with a phase-ON locked version of the neuO gene, resulting in constitutive O-acetylation of the K1 capsule. The phase-variable rate of neuO in MS7163 was measured by fragment analysis at 93% phase-ON with 21 heptanucleotide repeats. NeuO acetylated the K1 sialic acid into O-acetylated forms, including Neu5,7Ac2, Neu5,8Ac2, and Neu5,9Ac2. We demonstrate that O-acetylation increased survival of K1 UPEC to lytic K1 phage and human neutrophils, yet increased susceptibility to human serum. O-acetylation of the K1 capsule did not influence bladder colonization in a murine model of urinary tract infection. Overall, we hypothesize that phase-variable O-acetylation is a niche-specific adaptive mechanism that enhances survival of UPEC in different ways, including protection against K1 phage and resistance to neutrophil-mediated killing.IMPORTANCEThe K1 polysialic acid capsule is a key virulence factor of uropathogenic Escherichia coli (UPEC). A subset of K1 UPEC possesses the phage-encoded phase-variable neuO gene, which mediates O-acetylation of the capsule. However, the prevalence, phase dynamics, and biological consequences of this modification remain to be fully elucidated. Here, we show that the neuO gene exhibits variable distribution among K1 UPEC, with a high prevalence in the global ST95 clone and evidence for active phase switching. We further demonstrate that K1 O-acetylation confers resistance to phage and neutrophil killing, suggesting a role associated with enhanced survival in infection and environmental settings.

Methanol, a renewable non-food C1 substrate, holds great promise as a feedstock for sustainable biomanufacturing and carbon neutral production. However, its industrial application is hindered by low methanol assimilation efficiency in most microbes. Recent advances in synthetic biology and metabolic engineering have enabled the development of methylotrophic microbial cell factories through strategies including building efficient methanol-utilizing pathways, engineering methanol dehydrogenase for enhanced oxidation efficiency, and optimizing redox balance via cofactor utilization. Additionally, approaches such as mitigating the accumulation of toxic metabolites and adaptive laboratory evolution have been adopted to improve the robustness of synthetic methylotrophs. This review summarizes these innovations and provides a blueprint for rationally designing high-performance microbial platforms to facilitate industrial methanol utilization and advance sustainable development.

Bacteria localize proteins to distinct subcellular locations, including chemoreceptors, which frequently localize to the bacterial pole. Although some polarity-promoting mechanisms have been described, many chemoreceptors lack clear routes to becoming polar. TlpD of the bacterial pathogen Helicobacter pylori is one such protein. This cytoplasmic chemoreceptor localizes to the pole in a manner that is independent of the other chemoreceptors. In this work, we evaluated the role of TlpD domains in its function. Truncated proteins were created that lacked different amounts of the N- or C-termini and expressed in H. pylori in place of native tlpD or as the sole chemoreceptor. These TlpD variants were examined for their presence and abundance, protein localization, association with chemotaxis signaling proteins, and effect on motility. TlpD that lacked any portion of the N-terminal 104 amino acids produced low to no amounts of detectable protein. In contrast, TlpD was detectable with loss of the C-terminal 45 amino acids. TlpD lacking the last 45 amino acids (TlpD∆C4) preserved the ability to interact with CheW and CheV proteins based on bacterial two-hybrid analysis, but was unable to localize to the pole either on their own or in the presence of other chemoreceptors. TlpD∆C4 was found to be diffuse in the cytoplasm and interacted with CheV1, CheV2, and CheV3 at this location but not with CheW. TlpD∆C4 did not confer chemotactic abilities in soft agar chemotaxis assays. These findings suggest the C-terminal end of TlpD plays a previously unappreciated role in promoting TlpD polar localization and function.IMPORTANCEBacteria place their proteins in specific locations that are required for the proteins to function, including the bacterial pole. How the bacterial cell identifies which proteins go to the pole is not fully understood. In this work, we dissect parts of a protein called TlpD that naturally goes to the pole. We find that mutants lacking one end of TlpD lose their polar placement, but retain other abilities. TlpD allows directed motility known as chemotaxis. This ability is critical for infection in Helicobacter pylori and numerous other pathogens. When TlpD loses its polar placement, the protein no longer functions for chemotaxis, laying the foundation for future studies that can dissect how this segment promotes function and eventually translate into therapies for H. pylori infection.

Pseudomonas aeruginosa (Pa) is an important opportunistic pathogen that has many virulence factors expressed in a coordinated manner to cause infection. The PilY1 protein is a component of the type IV pili (T4P) but has additional signaling functions outside of the pilus. A major function of PilY1 is to prevent the AlgZ/R two-component system from functioning. However, the complete effects of PilY1 signaling through the control of the AlgZ/R or other regulatory systems are not well understood. In this study, we determine new genes controlled by the AlgZ/R system that are impacted by PilY1. We discovered that PilY1 impacts cAMP by preventing the AlgZ/R system from activating the major adenylate cyclase, cyaB. PilY1 also prevents the AlgZ/R system from activating itself and a putative c-di-GMP phosphodiesterase, PA4781. Furthermore, PilY1 functions in different P. aeruginosa strains, including mucoid strains. Surprisingly, bacterial survival in the lung, liver, and blood did not require PilY1. Overall, these findings suggest that PilY1 is critical for preventing AlgZ/R activity to prevent improper second messenger production. This work identifies new AlgZ/R targets and implicates the AlgZ/R system in the control of cAMP in P. aeruginosa. This work identifies a possible way to use PilY1 to prevent the expression of the major adenylate cyclase using PilY1 which could be used to impact P. aeruginosa virulence.IMPORTANCEUnderstanding PilY1 signaling is important for elucidating how P. aeruginosa adapts to different environments. A major function for PilY1 is to interfere with the AlgZ/R two-component system. Here, we use transcriptomics to determine genes PilY1 affects through the AlgZ/R system. We identified new AlgZ/R targets and established a mechanism for impacting cAMP levels. Our findings further our understanding of PilY1 and the AlgZ/R system and suggest a possible way that PilY1 might be used to prevent cAMP from increasing in P. aeruginosa.

Menstrual toxic shock syndrome (mTSS) is a rare yet life-threatening disease that is caused when the opportunistic pathogenic bacterium Staphylococcus aureus releases the toxic shock syndrome toxin 1 (TSST-1) superantigen, which triggers systemic inflammation. The main risk factor for mTSS is prolonged use of intravaginal products, which facilitates S. aureus growth and TSST-1 production in menstrual blood. However, some studies suggest that the vaginal microbiota may also play a role in mTSS occurrence. A previous study reported that the presence of S. aureus in menstrual fluids was correlated with the simultaneous presence of Candida species, particularly C. albicans. Here, we assessed the potential involvement of C. albicans in the stimulation of TSST-1 production by incubating S. aureus strains with C. albicans culture supernatants and measuring TSST-1 production. We found that the growth of C. albicans depleted the available glucose, thereby alleviating the CcpA-mediated repression of the tst gene, which encodes TSST-1, and enabling activation of its expression by SaeRS activity. These results highlight the importance of vaginal microbiota and nutrient availability with regard to the regulation of S. aureus virulence in the context of mTSS.IMPORTANCEMenstrual toxic shock syndrome (mTSS) is a life-threatening disease caused by toxic shock syndrome toxin 1 (TSST-1)-producing strains of Staphylococcus aureus. While tampons are a known risk factor, the vaginal microbiota may also increase mTSS risk. Our findings reveal that the yeast Candida albicans, a frequent colonizer of the vaginal mucosa, stimulates TSST-1 production of S. aureus by depleting glucose, a key regulator of tst gene expression. This study highlights how C. albicans, which is part of the vaginal microbiota, can amplify S. aureus virulence through metabolic interactions. These findings may also carry clinical implications by identifying vaginal colonization with C. albicans as a potential biomarker for heightened mTSS susceptibility, specifically in individuals harboring TSST-1-producing strains of S. aureus.

Despite decades of investigation into bacterial pathogens, the conditions met by intracellular bacteria are still unclear. These conditions can include access to nutrients, such as amino acids, and exposure to toxic compounds, like copper. To investigate the ability of Brucella abortus, a facultative intracellular pathogen responsible for a major zoonosis, to cope with copper, we performed a Tn-seq analysis to identify copper-sensitive mutants. Unexpectedly, we realized that classical copper resistance systems (involving CopA and CueO homologs) do not appear to be robustly needed, while histidine and purine biosynthesis pathways are crucial to cope with copper. We show that hisA, hisB, hisC, and hisD mutants are auxotrophic for histidine and sensitive to copper. This suggests that the reported attenuation of his mutants in macrophages could be based on auxotrophy and/or copper sensitivity. Therefore, we generated suppressor strains with a restored resistance to copper for hisC, but still auxotrophs for histidine. Our data suggest that this suppression is due to the overproduction of a homolog of OppA, a periplasmic oligopeptide-binding protein. Analysis of these suppressors shows that the absence of histidine biosynthesis capacity, and not copper sensitivity, is required for optimal growth of B. abortus in macrophages.IMPORTANCEInvestigating conditions in which intracellular bacteria grow inside host cells is challenging and often involves the characterization of attenuated bacterial mutants obtained by screening. But a single mutant can display two different phenotypes related to intracellular conditions. It was the case for histidine auxotrophs of Brucella abortus, an important zoonotic pathogen. These histidine auxotrophs are attenuated in a macrophage cell line, and they are also sensitive to copper stress. Using a suppressor strain still auxotroph for histidine but with an improved resistance to copper, we show that histidine auxotrophy, and not sensitivity to copper excess, is the main cause of attenuation in the conditions tested here.

Caulobacter species are common residents of soil and aquatic ecosystems, where bioavailable iron is often extremely limited. Like other diderm bacteria, Caulobacter crescentus can acquire Fe(III) via outer-membrane TonB-dependent transporters (TBDTs) that recognize and import ferric siderophore complexes. Although C. crescentus is not known to synthesize siderophores, it encodes multiple TBDTs that are transcriptionally regulated by the ferric uptake repressor (Fur), suggesting it acquires iron by scavenging xenosiderophores produced by neighboring microbes. To identify C. crescentus genes required for xenosiderophore utilization, we developed a barcoded transposon screen using ferrioxamine B (FXB), a hydroxamate-family siderophore produced by soil actinomycetes, as a model substrate. This screen identified hiuABC, a conserved, Fur-regulated operon that supports FXB-dependent iron acquisition. We provide evidence that hiuA encodes the primary TBDT responsible for uptake of ferrioxamines and ferrichrome (FC), structurally distinct members of the hydroxamate siderophore family. hiuB encodes a PepSY-domain protein with structural similarity to Pseudomonas aeruginosa FoxB, a known periplasmic ferri-siderophore reductase. hiuC encodes a small, hypothetical membrane protein predicted to form a functional complex with HiuB in the inner membrane. Both hiuB and hiuC are required for utilization of FXB and ferrioxamine E, indicating a shared role in iron acquisition from ferrioxamines. Surprisingly, utilization of FC as an iron source required hiuB but not hiuC, suggesting a substrate-specific role for HiuC in ferri-siderophore processing. We conclude that the conserved hiuABC operon encodes a set of proteins that enable bacteria to acquire iron from structurally diverse hydroxamate-family siderophores.IMPORTANCEIron is often a limiting nutrient due to its poor solubility in the presence of oxygen. To overcome this, some microbes produce specialized molecules known as siderophores, which tightly bind and solubilize iron, facilitating its uptake into the cell. Caulobacter species are common in freshwater, marine, and soil environments, and there is emerging evidence that they play important roles in plant-associated microbial communities. Here, we report the discovery of a three-gene system that allows Caulobacter crescentus to acquire iron from a set of siderophores produced by select soil bacteria and fungi. We define functional roles for each protein component of this system, which informs a mechanism by which Caulobacter can pirate iron-scavenging molecules produced by its neighbors.

Mycobacterium abscessus (Mab), a rapidly growing mycobacterial species with intrinsic and acquired resistance to multiple antibiotics, is an emerging public health concern. The rise in clinical cases of treatment-refractory infections of M. abscessus has propelled its research toward novel therapeutic approaches. The number of publications entitled "Mycobacterium abscessus" has increased by ~300% over the last decade, of which the majority of studies exploring the fundamental biology and pathogenesis of Mab have used the reference strain ATCC19977. However, whole-genome sequence analyses, combined with transposon-seq based functional genomics, reveal an open pan-genome with significant variations in the essential genes across ATCC19977 and clinical isolates. These new discoveries demand a careful selection of strains and growth conditions in experimental design. In this minireview, we discuss these challenges and propose a framework for future M. abscessus studies in silico, including a new web-based resource for pangenome analysis, in vitro, and in animal models.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes both acute and chronic infections in vulnerable populations. Treatment of P. aeruginosa infections is increasingly challenging due to multi-drug resistance, and biofilm formation during infection further increases antibiotic tolerance. Iron, which is sequestered by the host innate immune system, is also a key nutrient that is required for P. aeruginosa biofilm formation. The iron-responsive PrrF small regulatory RNAs (sRNAs) are key to P. aeruginosa's iron starvation response, promote the production of the Pseudomonas quinolone signal (PQS) quorum sensing molecule, and are required for virulence in murine lung infection. Prior work showed that the PrrF sRNAs are dispensable for biofilm formation; however, these studies were performed using flow-cell biofilms grown at room temperature. Here, we demonstrate a temperature dependency for PrrF in P. aeruginosa biofilm formation: the genes for these sRNAs are required for optimal biofilm formation at 37°C but not 25°C. We further show that a ∆pqsA mutant, which lacks production of PQS and related metabolites, phenocopies the ∆prrF mutant. These studies demonstrate the importance of the PrrF sRNAs in P. aeruginosa biofilm formation at body temperature and reveal a previously underappreciated role of temperature in iron homeostasis and P. aeruginosa biofilm physiology.IMPORTANCEBiofilm formation is a critical virulence trait for many microbial pathogens that confers tolerance to the host immune system and antimicrobials. Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms resulting in treatment failure. Iron is a known requirement for P. aeruginosa biofilm formation, yet the precise role of iron in biofilm physiology remains unclear. Here, we show that temperature alters the requirement for the PrrF small regulatory RNAs, key components of P. aeruginosa's iron starvation response, for biofilm formation. Specifically, PrrF is required for the optimal formation of flow-cell biofilms at 37°C but not at 25°C, yet most flow-cell biofilm studies are conducted at 25°C. These results demonstrate a previously underappreciated role of temperature in P. aeruginosa biofilm physiology.

Yersinia pestis is a gram-negative bacterium and the causative agent of plague. The Y. pestis virulence factor plasminogen activator protease (Pla) is an outer membrane aspartic protease that facilitates the dissemination of bacteria from the site of inoculation to deeper tissue during bubonic plague. During pneumonic plague, Pla acts as an adhesin, which contributes to the suppression of early innate immune responses in the lungs, and as a protease that aids in resisting bacterial killing by neutrophils. Two-component regulatory systems (TCSs) are involved in bacterial adaptation to environmental stressors such as changes in pH, changes in ion concentrations, and the presence of cationic antimicrobial peptides. TCSs consist of a membrane-bound sensor kinase that detects environmental stressors and activates a response regulator to coordinately alter gene expression. The PhoP/PhoQ TCS regulates virulence factors and known Pla homologs in a variety of gram-negative pathogenic bacteria including Escherichia coli and Salmonella species. In the work described here, we evaluate whether pla is regulated by PhoP/PhoQ in Y. pestis. We identify a putative PhoP-binding site within the -10 box and the +1 transcription start site of pla that is bound by recombinant PhoP. Surprisingly, we show that the expression of pla is suppressed by PhoP/PhoQ under a variety of physiologically relevant PhoP/PhoQ-inducing conditions that are expected to be encountered during infection. This work demonstrates the regulation of an essential Y. pestis virulence factor by the PhoP/PhoQ TCS for the first time and highlights the importance of tightly regulating virulence factors that function as proteases.IMPORTANCEYersinia pestis causes plague, a highly lethal infection that results from inoculation via an infected flea (bubonic plague) or inhalation of contaminated respiratory droplets via person-to-person transmission (pneumonic plague). The plasminogen activator protease (Pla) is a critical Y. pestis virulence factor that is essential to the progression of infection via either route of inoculation. In this work, we show for the first time that the well-established two-component regulatory system PhoP/PhoQ regulates the expression of pla. Under conditions found during mammalian infection, PhoP/PhoQ suppresses pla expression, presumably to limit aberrant cleavage of Pla substrates during the critical early stages of infection. These results show interaction between two key virulence loci for the first time, and shed light on the regulation of a critical Y. pestis virulence determinant.