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Mycobacterium abscessus (Mab) is a clinically significant pathogen and a highly genetically diverse species due to its large accessory genome. The functional consequence of this diversity remains unknown mainly because, to date, functional genomic studies in Mab have been primarily performed on reference strains. Given the growing public health threat of Mab infections, understanding the functional genomic differences among Mab clinical isolates can provide more insight into how its genetic diversity influences gene essentiality, clinically relevant phenotypes, and importantly, potential drug targets. To determine the functional genomic diversity among Mab strains, we conducted transposon-sequencing (TnSeq) on 21 genetically diverse clinical isolates, including 15 M. abscessus subsp. abscessus isolates and 6 M. abscessus subsp. massiliense isolates, cataloging all the essential and non-essential genes in each strain. Pan-genome analysis revealed a core set of 3,845 genes and a large accessory genome of 11,507. We identified 259 core essential genes across the 21 clinical isolates and 425 differentially required genes, representing ~10% of the Mab core genome. We also identified genes whose requirements were subspecies, lineage, and isolate-specific. Finally, by correlating TnSeq profiles, we identified 19 previously uncharacterized genetic networks in Mab. Altogether, we find that Mab clinical isolates are not only genetically diverse but functionally diverse as well.

Importance: This study investigates the genetic diversity of Mycobacterium abscessus (Mab), a bacteria known for causing difficult-to-treat infections. Researchers performed transposon-sequencing (TnSeq) on 21 different clinical isolates of Mab to identify essential and non-essential genes in each strain. Through this analysis, they identified core genes required for growth across all strains. Interestingly, they also identified genes whose requirement for growth or "essentiality" were subspecies, lineage, and isolate-specific. This study reveals that Mab's genetic diversity translates into significant functional differences among clinical isolates. Insights from this paper lay essential groundwork for future studies exploring the biological and clinical implications of genetic diversity in Mab clinical isolates. Understanding this diversity could guide targeted therapies and offer new insights into managing infections caused by Mab, a growing public health concern.

Metatranscriptomics is uncovering more and more diverse families of viruses with RNA genomes comprising the viral kingdom Orthornavirae in the realm Riboviria. Thorough protein annotation and comparison are essential to get insights into the functions of viral proteins and virus evolution. In addition to sequence- and hmm profile‑based methods, protein structure comparison adds a powerful tool to uncover protein functions and relationships. We constructed an Orthornavirae "structurome" consisting of already annotated as well as unannotated ("dark matter") proteins and domains encoded in viral genomes. We used protein structure modeling and similarity searches to illuminate the remaining dark matter in hundreds of thousands of orthornavirus genomes. The vast majority of the dark matter domains showed either "generic" folds, such as single α-helices, or no high confidence structure predictions. Nevertheless, a variety of lineage-specific globular domains that were new either to orthornaviruses in general or to particular virus families were identified within the proteomic dark matter of orthornaviruses, including several predicted nucleic acid-binding domains and nucleases. In addition, we identified a case of exaptation of a cellular nucleoside monophosphate kinase as an RNA-binding protein in several virus families. Notwithstanding the continuing discovery of numerous orthornaviruses, it appears that all the protein domains conserved in large groups of viruses have already been identified. The rest of the viral proteome seems to be dominated by poorly structured domains including intrinsically disordered ones that likely mediate specific virus-host interactions.

Importance: Advanced methods for protein structure prediction, such as AlphaFold2, greatly expand our capability to identify protein domains and infer their likely functions and evolutionary relationships. This is particularly pertinent for proteins encoded by viruses that are known to evolve rapidly and as a result often cannot be adequately characterized by analysis of the protein sequences. We performed an exhaustive structure prediction and comparative analysis for uncharacterized proteins and domains ("dark matter") encoded by viruses with RNA genomes. The results show the dark matter of RNA virus proteome consists mostly of disordered and all-α-helical domains that cannot be readily assigned a specific function and that likely mediate various interactions between viral proteins and between viral and host proteins. The great majority of globular proteins and domains of RNA viruses are already known although we identified several unexpected domains represented in individual viral families.

Rickettsia parkeri is an obligate intracellular, tick-borne bacterial pathogen that can cause eschar-associated rickettsiosis in humans. R. parkeri invades host cells, escapes from vacuoles into the cytosol, and undergoes two independent modes of actin-based motility mediated by effectors RickA or Sca2. Actin-based motility of R. parkeri enables bacteria to enter protrusions of the host cell plasma membrane that are engulfed by neighboring host cells. However, whether and how RickA and Sca2 independently contribute to cell-to-cell spread in vitro or pathogenicity in vivo has been unclear. Using live cell imaging of rickA::Tn and sca2::Tn mutants, we discovered both RickA and Sca2 contribute to different modes of cell-to-cell spread. Compared with Sca2-spread, RickA-spread involves the formation of longer protrusions that exhibit larger fluctuations in length and take a longer time to be engulfed into neighboring cells. We further compared the roles of RickA and Sca2 in vivo following intradermal (i.d.) infection of Ifnar1^-/-; Ifngr1^-/- mice carrying knockout mutations in the genes encoding the receptors for IFN-I (Ifnar1) and IFN-γ (Ifngr1), which exhibit eschars and succumb to infection with wild-type (WT) R. parkeri. We observed that RickA is important for severe eschar formation, whereas Sca2 contributes to larger foci of infection in the skin and dissemination from the skin to the internal organs. Our results suggest that actin-based motility effectors RickA and Sca2 drive two distinct forms of cell-to-cell spread and contribute differently to pathogenicity in the mammalian host.IMPORTANCERickettsia parkeri, a bacterium in the spotted fever group of Rickettsia species, can be transmitted from ticks to humans, leading to symptoms including fever, rash, muscle aches, and a lesion at the site of the tick bite. During Rickettsia parkeri infection, bacteria invade cells within the animal host, proliferate in the host cell's cytosol, move using a process called actin-based motility, and spread to neighboring host cells. Rickettsia parkeri is unusual in having two bacterial proteins that mediate actin-based motility. The significance of our research is to reveal that each of these bacterial actin-based motility proteins contributes differently to spread between cells and to the signs of infection in a mouse model of spotted fever disease. Our results are important for understanding the contribution of actin-based motility to mammalian infection by Rickettsia parkeri as well as to infection by other bacterial and viral pathogens that require this process to spread between cells and cause disease.

The KREMEN1 (KRM1) protein is a cellular receptor for multiple enteroviruses that cause hand, foot, and mouth disease (HFMD), including coxsackievirus CVA2, CVA3, CVA4, CVA5, CVA6, CVA10, and CVA12. The molecular basis for the broad recognition of these viruses by the KRM1 receptor remains unclear. Here, we report the indispensable role of the completely conserved VP2 capsid protein residue K140 (designated K2140) in mediating receptor recognition and infection by CVA10 and other KRM1-dependent enteroviruses. Residue K2140 not only facilitates receptor recognition, cell attachment, and infection of CVA10 but also contributes to CVA10 pathogenicity in vivo. Notably, residue K2140 is completely conserved in all strains of the KRM1-dependent enteroviruses. Mutational analysis confirms the importance of K2140 for infection by CVA2-CVA6, and CVA12. Moreover, CVA8, an enterovirus for which the cellular receptor has not yet been identified, also possesses the conserved K2140 residue. We experimentally demonstrate that CVA8 utilizes KRM1 as its receptor, with K2140 being essential for viral infection. Additionally, residue D90 of KRM1 engages with residue K2140 and plays a crucial role in KRM1-mediated enterovirus infections. Collectively, our findings underscore the significance of the absolutely conserved K2140 residue in receptor interactions and infection of all KRM1-binding enteroviruses, providing novel insights into the molecular basis of enterovirus infection and informing the development of broad-spectrum therapies against HFMD.

Importance: Hand, foot, and mouth disease (HFMD) annually affects millions of children worldwide. HFMD is caused by various enteroviruses, such as coxsackieviruses CVA6, CVA16, CVA10, and enterovirus 71 (EV-A71). Licensed inactivated EV-A71 vaccines do not provide cross-protection against other enteroviruses. There are no drugs specifically for HFMD. KREMEN1 (KRM1) serves as the cellular receptor for many HFMD-related enteroviruses, including CVA2-CVA6, CVA10, and CVA12. However, the molecular basis for broad recognition of these enteroviruses by the KRM1 receptor remains elusive. Here, we report that VP2 residue K140 (K2140) is completely conserved among all KRM1-dependent enteroviruses and is essential for virus-receptor binding and viral infection by interacting with residue D90 of KRM1. Overall, our findings provide a deeper understanding of the molecular basis of KRM1-dependent enterovirus infection in vitro and in vivo and may contribute to the development of broad-spectrum anti-enterovirus vaccines and treatments.

Two-component signal transduction systems (TCSs) are nearly ubiquitous across bacterial species and enable bacteria to sense and respond to specific cues for environmental adaptation. The Campylobacter jejuni BumSR TCS is unusual in that the BumS sensor exclusively functions as a phosphatase rather than a kinase to control phosphorylated levels of its cognate BumR response regulator (P-BumR). We previously found that BumSR directs a response to the short-chain fatty acid butyrate generated by resident microbiota so that C. jejuni identifies ideal lower intestinal niches in avian and human hosts for colonization. However, butyrate is an indirect cue for BumS and did not inhibit in vitro BumS phosphatase activity for P-BumR. In this work, we expanded the repertoire of lower intestinal metabolites that are cues sensed by BumS that modulate the expression of genes required for colonization to include the branched short-chain fatty acids isobutyrate and isovalerate. Unlike butyrate, isobutyrate and isovalerate inhibited in vitro BumS phosphatase activity for P-BumR, indicating that these metabolites are direct cues for BumS. Isobutyrate and isovalerate reduced the thermostability of BumS and caused a reorganization of protein structure to suggest how sensing these cues inhibits phosphatase activity. We also identified residues in the BumS sensory domain required to detect isobutyrate, isovalerate, and butyrate and for optimal colonization of hosts to reveal how gut bacteria can recognize these intestinal metabolites. Our work reveals how this unusual bacterial sensor phosphatase senses a repertoire of intestinal metabolites and how cues alter BumSR signal transduction to influence C. jejuni colonization of hosts.IMPORTANCETCSs are prevalent in many bacteria, but the cues sensed by each are not actually known for many of these systems. Microbiota-generated butyrate in human and avian hosts is detected by the Campylobacter jejuni BumS sensor phosphatase so that the bacterium identifies ideal lower intestinal niches for colonization. However, BumS only indirectly senses butyrate to inhibit dephosphorylation of its cognate BumR response regulator. Here, we expanded the repertoire of cues sensed by BumS to the branched-short chain fatty acids isobutyrate and isovalerate that are also abundant in the lower intestines. Both isobutyrate and isovalerate are potent, direct cues for BumS, whereas butyrate is an indirect cue. Leveraging isobutyrate and isovalerate as direct cues, we reveal BumS structure is altered upon cue detection to inhibit its phosphatase activity. We provide an understanding of the mechanics of an unusual mode of signal transduction executed by BumSR and other bacterial sensor phosphatase-driven TCSs.

Fusobacterium nucleatum is a human pathogen associated with intestinal conditions including colorectal cancer. Screening for gut-derived strains that exhibit anti-F. nucleatum activity in vitro revealed Streptococcus salivarius DPC6487 as a strain of interest. Whole-genome sequencing of S. salivarius DPC6487 identified a nisin operon with a novel structural variant designated nisin G. The structural nisin G peptide differs from the prototypical nisin A with respect to seven amino acids (Ile4Tyr, Ala15Val, Gly18Ala, Asn20His, Met21Leu, His27Asn, and His31Ile), including differences that have not previously been associated with a natural nisin variant. The nisin G gene cluster consists of nsgGEFABTCPRK with transposases encoded between the nisin G structural gene (nsgA) and nsgF, notably lacking an equivalent to the nisI immunity determinant. S. salivarius DPC6487 exhibited a narrower spectrum of activity in vitro compared to the nisin A-producing Lactococcus lactis NZ9700. Nisin G-producing S. salivarius DPC6487 demonstrated the ability to control F. nucleatum DSM15643 in an ex vivo model colonic environment while exerting minimal impact on the surrounding microbiota. The production of this bacteriocin by a gut-derived S. salivarius, its narrow-spectrum activity, and its anti-F. nucleatum activity in a model colonic environment indicates that this strain merits further attention with a view to harnessing its probiotic potential.IMPORTANCEFusobacterium nucleatum is a human pathogen associated with intestinal conditions, including colorectal cancer, making it a potentially important therapeutic target. Bacteriocin-producing probiotic bacteria demonstrate the potential to target disease-associated taxa in situ in the gut. A gut-derived strain Streptococcus salivarius DPC6487 was found to demonstrate anti-F. nucleatum activity, which was attributable to a gene encoding a novel nisin variant designated nisin G. Nisin G-producing S. salivarius DPC6487 demonstrated the ability to control an infection of F. nucleatum in a simulated model of the human distal colon while exerting minimal impact on the surrounding microbiota. Here, we describe this nisin variant produced by S. salivarius, a species that is frequently a focus for probiotic development. The production of nisin G by a gut-derived S. salivarius, its narrow-spectrum activity against F. nucleatum, and its anti-F. nucleatum activity in a model colonic environment warrants further research to determine its probiotic-related applications.

Plasmodium parasites have a complex life cycle that transitions between mosquito and mammalian hosts, and undergo continuous cellular remodeling to adapt to various drastic environments. Following hepatocyte invasion, the parasite discards superfluous organelles for intracellular replication, and the remnant organelles undergo extensive branching and mature into hepatic merozoites. Autophagy is a ubiquitous eukaryotic process that permits the recycling of intracellular components. Here, we show that the Plasmodium berghei autophagy-related E1-like enzyme Atg7 is expressed in the blood, sporozoites, and liver stages, localized to the parasite cytosol, and is essential for the localization of Atg8 on the membrane and the development of parasite blood and liver forms. We found that depleting Atg7 abolishes Atg8 lipidation, exocytosis of micronemes, organelle biogenesis, and the formation of merozoites during liver-stage development. Overall, this study establishes the essential functions of Atg7 in Plasmodium blood and liver stages, and highlights its role in maintaining the parasite's cellular homeostasis and organelle biogenesis.IMPORTANCEThe malaria life cycle involves two hosts, mosquitoes and vertebrates. Plasmodium parasites undergo complex intracellular and extracellular stages during this transition. Here, we report that an autophagy-related E1-like enzyme Atg7 is required to conjugate Atg8 on the apicoplast membrane. Atg7 depletion in Plasmodium berghei resulted in the loss of Atg8 lipidation and multiple defects like clearance of micronemes, organelle biogenesis, and maturation of hepatic schizonts during liver-stage development. The essentiality of Plasmodium Atg7 in blood and liver stages suggests it is a prospective target for developing autophagy-specific inhibitors. These results highlight the importance of autophagy in malaria parasite development.

Campylobacter is a serious health threat because of the rapid progressive evolution of antimicrobial resistance and efficient transmission from zoonotic as well as human sources. Resistance to fluoroquinolones and macrolides is particularly concerning as this compromises the two most effective oral antibiotic agents currently available for human campylobacteriosis. Here, we report on the prevalence and worldwide distribution of the operon cmeRABC, which encodes an efflux pump conferring high levels of combined resistance to fluoroquinolones and macrolides in Campylobacter strains isolated from poultry (n = 75) and children (n = 177). These mutations were found to be highly prevalent in isolates from poultry (62.7%) and children (29.4%) in Iquitos, Peru. We investigated the population structure of genes in the cmeRABC operon and identified a potential genetic bottleneck for the cmeA and cmeB genes. While most cmeB alleles segregate by species, alleles associated with high resistance to fluoroquinolones and macrolides were found in both Campylobacter jejuni and Campylobacter coli. We inferred that the likely ancestry of these alleles was from C. jejuni and was later acquired by C. coli through recombination. Publicly accessible global genomic data from 16,120 Campylobacter genomes identified these mutations in approximately 6% of C. jejuni and C. coli isolates globally, with higher prevalence in samples from poultry in many countries, including Peru. Our findings suggest that these extensively drug-resistant Campylobacter strains originated from C. jejuni in poultry.IMPORTANCEAntimicrobial resistance in Campylobacter is a growing public health concern, driven by the rapid evolution and zoonotic transmission of resistant strains. This study focuses on mutations in the cmeABC efflux pump, which confer high resistance to fluoroquinolones and macrolides, the two most effective oral antibiotics for human campylobacteriosis. By analyzing genomes from poultry and children in Iquitos, Peru, as well as global genomic data sets, we identified a significant prevalence of these resistance-associated mutations, particularly in poultry and children. Our findings suggest that these mutations originated in Campylobacter jejuni and spread to C. coli through recombination. Globally, these mutations are found in approximately 6% of isolates, with higher prevalence in poultry in multiple countries. This research underscores the critical role of genomic epidemiology in understanding the origins, evolution, and dissemination of antimicrobial resistance and highlights the need to address poultry as a reservoir for resistant Campylobacter.

The rapid increase in infections caused by the emerging fungal pathogen Candida auris is of global concern, and understanding its expansion is a priority. The phylogenetic diversity of the yeast is clustered in five major clades, among which clade III is particularly relevant, as most of its strains exhibit resistance to fluconazole, reducing the therapeutic alternatives and provoking outbreaks that are difficult to control. In this study, we have investigated the phylogenetic structure of clade III by analyzing a global collection of 566 genomes. We have identified three subgroups within clade III, among which two are genetically most closely related. Moreover, we have estimated the evolutionary rate of clade III to be 2.25e-7 s/s/y (2.87 changes per year). We found that one of these subgroups shows intrinsic resistance to fluconazole and is responsible for the majority of cases within this clade globally. We inferred that this subgroup may have originated around December 2010 (95% High Probability Density (HPD): April 2010-June 2011), and since then it has spread across continents, generating multiple large outbreaks, each with a unique pattern of transmission and dissemination. These results highlight the remarkable ability of the pathogen to adapt to its environment and its rapid global spread, underscoring the urgent need to address this epidemiological challenge effectively.IMPORTANCEThe number of cases affected by Candida auris has increased worryingly worldwide. Among the currently recognized clades, clade III has the highest proportion of fluconazole-resistant cases and is spreading very rapidly, causing large nosocomial outbreaks across the globe. By analyzing complete fungal genomes from around the world, we have confirmed the origin of this clade and unraveled its dispersal patterns in the early 2010s. This finding provides knowledge that may be helpful to the public health authorities for the control of the disease.

Legionella species evade degradation and proliferate within alveolar macrophages as an essential step for the manifestation of disease. However, most intracellular bacterial pathogens are restricted in neutrophils, which are the first line of innate immune defense against invading pathogens. Bacterial degradation within neutrophils is mediated by the fusion of microbicidal granules to pathogen-containing phagosomes and the generation of reactive oxygen species (ROS) by the phagocyte NADPH oxidase complex. Here, we show that human neutrophils fail to trigger microbicidal processes and, consequently, fail to restrict L. longbeachae. In addition, neutrophils infected with L. longbeachae fail to undergo a robust pro-inflammatory response, such as degranulation and IL-8 production. Here, we identify three strategies employed by L. longbeachae for evading restriction by neutrophils and inhibiting the neutrophil microbicidal response to other bacteria co-inhabiting in the same cell. First, L. longbeachae excludes the cytosolic and membrane-bound subunits of the phagocyte NADPH oxidase complex from its phagosomal membrane independent of the type 4 secretion system (T4SS). Consequently, infected neutrophils fail to generate robust ROS in response to L. longbeachae. Second, L. longbeachae impedes the fusion of azurophilic granules to its phagosome and the phagosomes of bacteria co-inhabiting the same cell through T4SS-independent mechanisms. Third, L. longbeachae protects phagosomes of co-inhabiting bacteria from degradation by ROS through a trans-acting T4SS-dependent mechanism. Collectively, we conclude that L. longbeachae evades restriction by human neutrophils via T4SS-independent mechanisms and utilizes trans-acting T4SS-dependent mechanisms for inhibition of neutrophil ROS generation throughout the cell cytosol.

Importance: Legionella longbeachae is commonly found in soil environments where it interacts with a wide variety of protist hosts and microbial competitors. Upon transmission to humans, L. longbeachae invades and replicates within alveolar macrophages, leading to the manifestation of pneumonia. In addition to alveolar macrophages, neutrophils are abundant immune cells acting as the first line of defense against invading pathogens. While most intracellular bacterial species are killed and degraded by neutrophils, we show that L. longbeachae evades degradation. The pathogen impairs the major neutrophils' microbicidal processes, including the fusion of microbicidal granules to the pathogen-containing vacuole. By inhibiting of assembly of the phagocyte NADPH oxidase complex, the pathogen blocks neutrophils from generating microbicide reactive oxygen species. Overall, L. longbeachae employs unique virulence strategies to evade the major microbicidal processes of neutrophils.