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Clostridioides difficile infections cause over 12,000 deaths and an estimated one billion dollars in healthcare costs annually in the United States. The cell membrane is an essential structure that is important for protection from the extracellular environment, signal transduction, and transport of nutrients. The polar membrane lipids of C. difficile are ~50% glycolipids, a higher percentage than most other organisms. The glycolipids of C. difficile consist of monohexosyldiradylglycerol (MHDRG) (~14%), dihexosyldiradylglycerol (DHDRG) (~15%), trihexosyldiradylglycerol (THDRG) (~5%), and a unique glycolipid aminohexosyl-hexosyldiradylglycerol (HNHDRG) (~16%). Previously, we found that HexSDF are required for the synthesis of HNHDRG. The enzymes required for the synthesis of MHDRG, DHDRG, and THDRG are not known. In this study, we identified the glycosyltransferases UgtA (CDR20291_0008), which is required for the synthesis of all glycolipids, and UgtB (CDR20291_1186), which is required for the synthesis of DHDRG and THDRG. We propose a model where UgtA synthesizes only MHDRG, HexSDF synthesize HNHDRG from MHDRG, and UgtB synthesizes DHDRG and potentially THDRG from MHDRG. We also report that glycolipids are important for critical cell functions, including sporulation, cell size and morphology, maintaining membrane fluidity, colony morphology, and resistance to some membrane-targeting antimicrobials.

Importance: Clostridioides difficile infections are the leading cause of healthcare-associated diarrhea. C. difficile poses a risk to public health due to its ability to form spores and cause recurrent infections. Glycolipids make up ~50% of the polar lipids in the C. difficile membrane, a higher percentage than other common pathogens and include a unique glycolipid not present in other organisms. Here, we identify glycosyltransferases required for the synthesis of glycolipids in C. difficile and demonstrate the important role glycolipids play in C. difficile physiology.

The field of secondary metabolism has greatly benefitted from computational advances in recent years. This has been particularly true for fungal natural product studies. Strides in genome mining have led to the identification of an extraordinary number of secondary metabolite biosynthetic gene clusters (BGCs) across the fungal Kingdom and metabologenomic platforms can group BGCs into gene cluster families and link them to initial chemical structures. Missing are computational applications focused on identifying BGC regulatory networks. Here, we applied the new online gene regulatory network resource, GRAsp (Gene Regulation of Aspergillus fumigatus), to identify unknown and unpredictable BGC trans-acting transcriptional/metabolite production modules. GRAsp correctly predicted a two-component regulatory module composed of the transcription factors (TFs), RogA (regulation of gliotoxin) and HsfA, which negatively regulate the gliotoxin BGC and are also involved in gliotoxin self-protection. RogA functions through the repression of gliZ, the pathway-specific gliotoxin TF, and HsfA functions by activating rogA expression. Furthermore, GRAsp identified TFs that regulate the production of two BGCs lacking pathway-specific TFs, the helvolic acid and fumitremorgin BGCs, respectively. Finally, the known TF, NsdD, was predicted and found to regulate the hexadehydroastechrome BGC. These advances highlight the power of inference algorithms to uncover unpredictable networks in specialized metabolite synthesis.IMPORTANCEToxic secondary metabolites are virulence factors of the opportunistic fungal pathogen Aspergillus fumigatus, yet the transcriptional networks regulating secondary metabolite production remain elusive. Uncovering novel regulators without any prior information is challenging. Computational programs have gained prominence in the field of secondary metabolite research due to their accuracy and ability to handle vast amounts of data, including DNA, RNA, and protein data. In this study, a newly developed online computer platform, Gene Regulation of A. fumigatus, was used to identify five regulators involved in the production of several A. fumigatus toxins, including gliotoxin, helvolic acid, fumitremorgin, and hexadehydroastechrome. This work illustrates the potential for discovering new trans-acting regulators and mechanisms of secondary metabolite regulation through the examination of computational gene regulatory networks.

White spot syndrome virus (WSSV) is a debilitating viral pathogen that poses a significant threat to the global crustacean farming industry. It has a wide host tropism because it uses several receptors to facilitate its attachment and entry. Thus far, not all the receptors have been identified. Here, we employed a BioID-based screening method to identify the Na⁺-K⁺-ATPase alpha subunit (PvATP1A) as a potential receptor in Penaeus vannamei. Although during the early stages of WSSV infection, PvATP1A was induced and underwent oligomerization, clustering, and internalization, knockdown of PvATP1A inhibited viral entry and replication. PvATP1A interacted with the WSSV envelope protein VP28 through its multiple extracellular regions, whereas synthetic PvATP1A extracellular region peptides blocked WSSV entry and replication. We showed that PvATP1A did not affect WSSV attachment but facilitated internalization via caveolin-mediated endocytosis and macropinocytosis. These findings provide a robust receptor screening approach that identified PvATP1A as an entry receptor for WSSV, presenting a novel target for the development of anti-WSSV therapeutics.

Importance: Cell surface receptors are crucial for mediating virus entry into host cells. Identification and characterization of virus receptors are fundamental yet challenging aspects of virology research. In this study, a BioID-based screening method was employed to identify the Na⁺-K⁺-ATPase alpha subunit (PvATP1A) as a potential receptor for white spot syndrome virus (WSSV) in the shrimp Penaeus vannamei. We demonstrated that PvATP1A interacted with the WSSV envelope protein VP28 via its multiple extracellular regions, thereby promoting viral internalization through caveolin-mediated endocytosis and macropinocytosis. Importantly, compared with previously identified WSSV receptors such as β-integrin, glucose transporter 1 (Glut1), and polymeric immunoglobulin receptor (pIgR), PvATP1A demonstrated significantly enhanced viral entry, indicating that PvATP1A is a crucial entry receptor of WSSV. This study not only presents a robust approach for screening virus receptors but also identifies PvATP1A as a promising target for the development of anti-WSSV therapeutics.

Capsule polysaccharide (CPS) is among the most important virulence factors of Klebsiella pneumoniae. Previous studies demonstrated that CPS plays multiple functional roles, but the mechanism by which this virulence factor enhances the survival fitness of K. pneumoniae remains unclear. In this work, we demonstrate that CPS is the main cellular component that not only elicits the host immune response to K. pneumoniae but also enables this pathogen to survive for a prolonged period under adverse environmental conditions. Consistently, our in vitro experiments suggest that CPS prevents K. pneumoniae from phagocytosis, rendering the encapsulated strain more difficult to be eradicated by the host. We also found that phagocytosis of K. pneumoniae is partially mediated by LOX-1, a scavenger receptor of the host, and that CPS may impede interaction between LOX-1 and this pathogenic bacteria, therefore reducing the phagocytosis process. These findings provide insights into the pathogenic mechanisms of this important clinical pathogen and should facilitate the design of new strategies to combat K. pneumoniae infections.

Importance: Klebsiella pneumoniae has become one of the most important clinical bacterial pathogens due to its evolution into hyperresistant and hypervirulent phenotypes. The mechanism of virulence of this pathogen is not well understood, particularly because it differs from other Enterobacteriaceae pathogens such as Escherichia coli and Salmonella. The capsule polysaccharide (CPS) of this pathogen is well recognized for contributing to the virulence of K. pneumoniae, but the exact mechanisms underlying its contribution are unclear. In this study, we demonstrated that CPS does not directly contribute to the host response; rather, it forms an external coat that blocks host recognition and prevents immune cells from binding to receptor proteins on K. pneumoniae, thus inhibiting phagocytosis, which makes it more challenging for the body to fight off infections. Understanding these mechanisms is vital for developing new treatments against K. pneumoniae infections, ultimately improving patient outcomes and public 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.

The healthy human infant gut microbiome undergoes stereotypical changes in taxonomic composition between birth and maturation to an adult-like stable state. During this time, extensive communication between microbiota and the host immune system contributes to health status later in life. Although there are many reported associations between microbiota compositional alterations and disease in adults, less is known about how microbiome development is altered in pediatric diseases. One pediatric disease linked to altered gut microbiota composition is cystic fibrosis (CF), a multi-organ genetic disease involving impaired chloride secretion across epithelia and heightened inflammation both in the gut and at other body sites. Here, we use shotgun metagenomics to profile the strain-level composition and developmental dynamics of the infant fecal microbiota from several CF and non-CF longitudinal cohorts spanning from birth to greater than 36 months of life. We identify a set of keystone species that define microbiota development in early life in non-CF infants but are missing or decreased in relative abundance in infants with CF, resulting in a delayed pattern of microbiota maturation, persistent entrenchment in a transitional developmental phase, and subsequent failure to attain an adult-like stable microbiota. Delayed maturation is strongly associated with cumulative antibiotic treatments, and we also detect the increased relative abundance of oral-derived bacteria and higher levels of fungi in infants with CF, features that are associated with decreased gut bacterial density. These findings suggest the potential for future directed therapies targeted at overcoming developmental delays in microbiota maturation for infants with CF.IMPORTANCEThe human gastrointestinal tract harbors a diversity of microbes that colonize upon birth and collectively contribute to host health throughout life. Infants with the disease cystic fibrosis (CF) harbor altered gut microbiota compared to non-CF counterparts, with lower levels of beneficial bacteria. How this altered population is established in infants with CF and how it develops over the first years of life is not well understood. By leveraging multiple large non-CF infant fecal metagenomic data sets and samples from a CF cohort collected prior to highly effective modulator therapy, we define microbiome maturation in infants up to 3 years of age. Our findings identify conserved age-diagnostic species in the non-CF infant microbiome that are diminished in abundance in CF counterparts that instead exhibit an enrichment of oral-derived bacteria and fungi associated with antibiotic exposure. Together, our study builds toward microbiota-targeted therapy to restore healthy microbiota dynamics in infants with CF.

All organisms produce an intracellular Zn²⁺-dependent enzyme, phosphomannose isomerase (PMI) or mannose-6 phosphate isomerase, that catalyzes the reversible conversion of mannose-6-phosphate and fructose-6-phosphate during sugar metabolism and polysaccharide biosynthesis. Unexpectedly, we discovered an additional PMI function in Borrelia burgdorferi, the pathogen of Lyme disease, where the enzyme is localized on the cell surface and binds to collagen IV-a host extracellular matrix component predominantly found in the skin. The AlphaFold 3-based structural model of B. burgdorferi PMI (BbPMI) retains the active site with tetrahedrally-coordinated Zn²⁺ seen in other PMIs of known structure, residing in an elongated crevice. Ligand docking shows that the crevice can accommodate the tip trisaccharide moiety of a glycosylated asparagine residue on the collagen IV 7S domain. Low doses of a well-known PMI benzoisothiazolone inhibitor impair the growth of diverse strains of B. burgdorferi in culture, but not other tested Gram-negative or Gram-positive pathogens. Borrelia cells are even more susceptible to several other structurally related benzoisothiazolone analogs. The passive transfer of anti-BbPMI antibodies in ticks can impact spirochete transmission to mice, while the treatment of collagen IV-containing murine skin with PMI inhibitors impairs spirochete infectivity. Taken together, these results highlight a newly discovered role for BbPMI in mediating host-pathogen interactions during the spirochete infectivity process. In turn, this discovery offers an opportunity for the development of a novel therapeutic strategy to combat Lyme disease by preventing the BbPMI interaction with its host receptor, collagen IV.

Importance: All organisms produce an intracellular enzyme, phosphomannose isomerase (PMI), that converts specific sugars during metabolism. Unexpectedly, we discovered an additional PMI function in Borrelia burgdorferi, the Lyme disease pathogen, where the enzyme is localized on the cell surface and binds to collagen IV-a host extracellular molecule mainly found in the skin. Low doses of PMI chemical inhibitors impair the growth of diverse strains of B. burgdorferi in culture, but not other tested bacterial pathogens. The passive transfer of anti-BbPMI antibodies in ticks can impact B. burgdorferi transmission to mice, while the treatment of collagen IV-containing murine skin with PMI inhibitors impairs infectivity. Taken together, these results highlight a newly discovered role for BbPMI in mediating host-pathogen interactions during infection. In turn, this discovery offers an opportunity for the development of a novel therapeutic strategy to combat Lyme disease by preventing BbPMI function and interaction with host collagen IV.

The central, mortality-associated hallmark of cancer is the process of metastasis. It is increasingly recognized that bacteria influence multiple facets of cancer progression, but the extent to which tumor microenvironment-associated bacteria control metastasis in cancer is poorly understood. To identify tumor-associated bacteria and their role in metastasis, we utilized established murine models of non-metastatic and metastatic breast tumors to identify bacteria capable of driving metastatic disease. We found several species of the Bacillus genus that were unique to metastatic tumors, and found that breast tumor cells cultured with a Bacillus bacterium isolated from metastatic tumors, Bacillus thermoamylovorans, produced nearly 3× the metastatic burden as control cells or cells cultured with bacteria from non-metastatic breast tumors. We then performed targeted metabolomics on tumor cells cultured with different bacterial species and found that B. thermoamylovorans differentially regulated tumor cell metabolite profiles compared to bacteria isolated from non-metastatic tumors. Using these bacteria, we performed de novo sequencing and tested for the presence of genes that were unique to the bacterium isolated from metastatic tumors in a patient population to provide a proof-of-concept for identifying how specific bacterial functions are associated with the metastatic process in cancer independent of bacterial species. Together, our data directly demonstrate the ability of specific bacteria to promote metastasis through interaction with cancer cells.

Importance: Metastasis is a major barrier to long-term survival for cancer patients, and therapeutic options for patients with aggressive, metastatic forms of breast cancer remain limited. It is therefore critical to understand the differences between non-metastatic and metastatic disease to identify potential methods for slowing or even stopping metastasis. In this work, we identify a bacterial species present with metastatic breast tumors capable of increasing the metastatic capabilities of tumor cells. We isolated and sequenced this bacteria, as well as a control species which failed to promote metastasis, and identified specific bacterial genes that were unique to the metastasis-promoting species. We tested for the presence of these bacterial genes in patient tumor samples and found they were more likely to be associated with mortality. We also identified enrichment of specific bacterial functions, providing insight into possible sources of bacteria-driven increases in the metastatic potential of multiple cancer types.

To survive in harsh natural environments, translation and mRNA metabolism must be tightly and coordinately controlled, as saving biological costs increases fitness. However, the roles of protein chaperones in this control system are unclear. This study proposes the novel aspect of the link between translation and mRNA metabolism, that is, the co-translational DnaJK chaperone activity is involved in changes in mRNA metabolism by RNase P. We found that the expression of proBA, which encodes proline biosynthetic enzymes, is regulated by ylxR(rnpM) through the proBA promoter. YlxR(RnpM), which is associated with RNase P, was also involved in the posttranscriptional regulation of proBA. To clarify this posttranscriptional regulation, we screened transposon (Tn)-inserted mutants for cells with low proB::lacZ expression and identified the DnaJK chaperone as a regulator of proB. To explore the possibility that the complex of YlxR(RnpM) and RNase P might work with DnaJK, we performed an epistatic analysis using the lacZ fusions, which revealed that the regulation of proB by DnaJK/YlxR(RnpM)/RNase P, that is, co-translational chaperone activity, controlled mRNA metabolism. RNA sequencing analysis of cells deficient in the RNA component of RNase P (rnpB) revealed that 261 genes were upregulated in the rnpB::Tn strain. Among them, we identified yoyD/yodF, besA, and epeXE, which were also under the control of DnaJK/YlxR(RnpM)/RNase P regulatory cascade. Finally, we performed yeast two-hybrid analysis using DnaK as bait and identified two genes, spoIVCA and nupG, whose expression was post-transcriptionally regulated by DnaJK but independent of YlxR(RnpM). These results suggest a broader role for posttranscriptional gene regulation by DnaJK.IMPORTANCEBacillus subtilis lacking the DnaJK chaperone has not been reported to exhibit a distinct phenotype. However, our study revealed proline-dependent growth in a minimal medium in the dnaJ::Tn strain. Inhibition of spoIVCA expression in this strain was identified as a probable cause of the sporulation deficiency in previous and current studies using a single cell-level analysis. We also observed posttranscriptional regulation of proBA by the DnaJK and YlxR(RnpM)/RNase P complex. LacZ analyses of proB::lacZ in different backgrounds suggested that the above regulation ultimately functions in mRNA metabolism. In DnaJK-deficient cells, the nascent peptide may be misfolded, and if DnaJK chaperone activity is lost, such a signal may be transferred to RNase P. Therefore, proBA mRNA may be degraded in an RNase P-dependent manner if the misfolding of the polypeptide translated from this mRNA is detected. This system is useful for reducing the biological costs of futile mRNA elongation.

Binding to cellular receptors initiates viral replication and dictates sites in the host infected by the virus. As illustrated by mammalian orthoreovirus (reovirus), viruses can bind several types of receptors using distinct capsid components to facilitate the viral entry steps of attachment, internalization, and disassembly. The outer of the two concentric capsids of reovirus virions is formed by four viral proteins, three of which bind receptors. These capsid-receptor interactions mediate stepwise entry of reovirus, dictate viral tropism in infected animals, and expand the viral host range. Engagement of independent receptors by different capsid proteins is a property of many pathogenic viruses and illustrates common themes of receptor use in viral entry and disease.