mBio最新文献

Peptidoglycan (PG)-modifying enzymes play a crucial role in cell wall remodeling, essential for growth and division. Cell wall degradation products are transported to the cytoplasm and recycled back in most gram-negative bacteria, and PG recycling is also linked to β-lactam resistance in many bacteria. Caulobacter crescentus is intrinsically resistant to β-lactams. Recently, it was shown that a soluble lytic transglycosylase, SdpA, is essential for β-lactam resistance. However, the precise role of SdpA in β-lactam resistance is unknown. This study investigated the PG recycling pathway and its role in antibiotic resistance in C. crescentus. Anhydromuropeptides generated by the action of lytic transglycosylases (LTs) are transported to the cytoplasm by the permease AmpG. C. crescentus encodes an ampG homolog, and deletion mutants of sdpA and ampG are sensitive to β-lactams. The ampG deletion mutant displays a significant accumulation of anhydromuropeptides in the periplasm of C. crescentus, demonstrating its essential role in PG recycling. While single knockout mutants of sdpA and ampG exhibit no growth defects, double-deletion mutants (∆sdpA∆ampG) exhibit severe growth and morphological defects. These double mutants also show enhanced sensitivity to β-lactams. Analysis of soluble muropeptides in wild-type (WT), ∆sdpA, and ∆ampG mutants revealed reduced levels of PG precursors (UDP-GlcNAc, UDP-MurNAc, and UDP-MurNAc-P5), suggesting that PG recycling products contribute toward de novo PG biosynthesis. Furthermore, supplementing the growth media with GlcNAc sugar enhanced the fitness of ∆sdpA and ∆ampG mutants under β-lactam stress. In conclusion, our study indicates that defects in PG recycling compromise cell wall biogenesis, leading to antibiotic sensitivity in C. crescentus.IMPORTANCEβ-lactam antibiotics target the peptidoglycan cell wall biosynthetic pathway in bacteria. In response to antibiotic pressures, bacteria have developed various resistance mechanisms. In many gram-negative species, cell wall degradation products are transported into the cytoplasm and induce the expression of β-lactamase enzymes. In this study, we investigated the cell wall recycling pathway and its role in antibiotic resistance in Caulobacter crescentus. Based on our data and prior studies, we propose that cell wall degradation products are utilized for the synthesis of peptidoglycan precursors in the cytoplasm. A deficiency in cell wall recycling leads to cell wall defects and increased antibiotic sensitivity in C. crescentus. These findings are crucial for understanding antibiotic resistance mechanisms in bacteria.

The bacterium Listeria monocytogenes (Lm) causes listeriosis in humans and ruminants. Acute lesions are predominantly infiltrated by polymorphonuclear neutrophils (PMNs), considered to be the efficient bactericidal arm of innate immunity. However, recent evidence suggests that PMNs cannot achieve antilisterial sterilizing immunity and that Lm may persist within PMNs. Despite this, interactions between PMNs and Lm remain poorly understood. In this study, we characterized the listericidal activity and interaction dynamics of bovine PMNs with Lm ex vivo. Phagocytosed Lm failed to escape into the PMN cytosol and was primarily targeted by phagolysosomal mechanisms. However, PMNs enabled prolonged intravacuolar survival of a resilient Lm subpopulation, largely as viable but non-culturable (VBNC) bacteria. This resilient Lm population could spread from PMNs to a cell line, resuscitate, and complete its canonical life cycle, thereby perpetuating the infection. Therefore, we identify PMNs as a mobile niche for Lm survival and provide evidence that PMNs harbor VBNC bacteria, potentially facilitating Lm dissemination within the host.

Importance: Listeria monocytogenes (Lm) is a significant foodborne pathogen responsible for high hospitalization rates in humans, especially vulnerable groups such as the elderly, pregnant women, and immunocompromised individuals. In animals like ruminants, Lm infection leads to severe disease manifestations, notably brainstem encephalitis. This study uncovers a novel mechanism by which bovine neutrophils (PMNs) harbor Lm in a viable but non-culturable (VBNC) state, enabling the bacteria to hide in the host. PMNs, traditionally viewed as bacteria killers, may serve as Trojan horses, allowing Lm to persist and spread within the host. This discovery has broad implications for understanding Lm's persistence, its role in recurrent infections, and the development of new therapeutic strategies targeting VBNC forms of Lm to improve treatment outcomes and disease control.

Fusobacterium nucleatum (Fn) is an oral commensal inhabiting the human gingival plaque that is rarely found in the gut. However, in colorectal cancer (CRC), Fn can be isolated from stool samples and detected in metagenomes. We hypothesized that ecological characteristics of the gut are altered by disease, enabling Fn to colonize. Multiple genomically distinct populations of Fn exist, but their ecological preferences are unstudied. We identified six well-separated populations in 133 Fn genomes and used simulated metagenomes to demonstrate sensitive detection of populations in human oral and gut metagenomes. In 9,560 samples from 11 studies, Fn population C2 animalis is elevated in gut metagenomes from CRC and Crohn's disease patients and is observed more frequently in CRC stool samples than in the gingiva. Polymorphum, the most prevalent gingival Fn population, is significantly increased in Crohn's stool samples; this effect was significantly stronger in male hosts than in female. We find polymorphum genomes are enriched for biosynthetic gene clusters and fluoride exporters, while C2 animalis are high in iron transporters. Fn populations thus associate with specific clinical and demographic phenotypes and harbor distinct functional features. Ecological differences in closely related groups of bacteria inform microbiome impacts on human health.

Importance: Fusobacterium nucleatum is a bacterium normally found in the gingiva. F. nucleatum generally does not colonize the healthy gut, but is observed in approximately a third of colorectal cancer (CRC) patient guts. F. nucleatum's presence in the gut during CRC has been linked to worse prognosis and increased tumor proliferation. Here, we describe the population structure of F. nucleatum in oral and gut microbiomes. We report substantial diversity in gene carriage among six distinct populations of F. nucleatum and identify population disease and body-site preferences. We find the C2 animalis population is more common in the CRC gut than in the gingiva and is enriched for iron transporters, which support gut colonization in known pathogens. We find that C2 animalis is also enriched in Crohn's disease and type 2 diabetes, suggesting ecological commonalities between the three diseases. Our work shows that closely related bacteria can have different associations with human physiology.

T follicular helper (Tfh) cells are crucial for B cell activation and subsequent antibody production. This functionality is influenced by surface markers such as CD40L, a costimulatory factor which promotes B cell activation, and CD57, which is a well-known marker of senescence. This study examined age-specific differences in Tfh cell function in Bangladeshi and American children. At age two, Bangladeshi children displayed impaired CD40L upregulation and significant CD57 downregulation upon stimulation. These patterns, not observed in American children of the same age, suggested an exhaustion-like phenotype potentially driven by environmental factors. Random forest and generalized estimating equations (GEE) modeling was used to analyze predictors of Tfh cell response to stimulation. Days since the last antibiotic treatment, total antibiotic treatments, diarrheal episodes, and malnutrition were identified as variables that significantly impacted the Tfh response to stimuli. To assess Tfh cell ability to promote antibody responses, we correlated Tfh functionality with antibody concentration post-vaccination and in response to infection with Cryptosporidium, an endemic apicomplexan parasite. Increased CD40L expression upon stimulation correlated positively with anti-Poliovirus type 2/3 neutralizing antibody and anti-Cp17 (a Cryptosporidium sporozoite antigen) IgA concentrations. In contrast, increased CD57 expression was significantly correlated with decreased anti-Cp17 IgA. This indicates that an activation-supportive phenotype (CD40L+) may be more effective in promoting immunity than a senescent phenotype (CD57+). Together, these findings suggest that early-life environmental exposures may program Tfh cell functionality, impacting immune response potential in settings with high pathogen exposure.

Importance: T follicular helper (Tfh) cells are upstream mediators that shape the humoral immune response to specific antigens. The generation of an effective memory response to infection is vital to prevent subsequent reinfections. However, in areas with high burdens of exposure to infections, such as the urban community from Bangladesh studied here, children are consistently exposed to inflammatory pathogens. Specific environmental exposures significantly influenced Tfh cell activation and senescence phenotypes. Additionally, Tfh cell responses correlated with antibody concentrations following vaccination or infection, indicating that environmental factors may play a critical role in shaping effective immunity in early childhood.

Type II toxin-antitoxin (TA) systems are widespread in prokaryotes. They consist of neighboring genes encoding two small proteins: a toxin that inhibits a critical cellular process and an antitoxin that binds to and neutralizes the toxin. The VapD nuclease and the VapX antitoxin comprise a type II TA system that contributes to the virulence of the human pathogen Haemophilus influenzae. We analyzed the diversity and evolution of VapD-like proteins. By examining loci adjacent to genes coding for VapD-like proteins, we identified two novel families of antitoxins, which we named VapY and VapW. VapD toxins cognate to novel antitoxins induce the SOS response when overproduced, suggesting they target cellular processes related to genomic DNA integrity, maintenance, or replication. Though VapY has no sequence similarity to VapX, they share the same SH3 fold characterized by the five anti-parallel β sheets that form a barrel. VapW is a homolog of VapD without conserved catalytic residues required for nuclease activity. The crystal structure of the VapD-VapW complex reveals that VapW lacks the dimerization interface essential for the catalytic activity of VapD but retains the second interaction interface that enables VapD hexamerization. This allows VapW to bind VapD in the same manner that VapD dimers bind to each other in hexamers. Thus, though the VapD catalytic cleft remains accessible in the VapD-VapW complex, VapW may disrupt VapD oligomerization. To our knowledge, VapWD provides a unique example of TA systems evolution when a toxin loses its activity and becomes an antitoxin to itself.

Importance: Genes encoding virulence-associated protein D (VapD) homologs are found in many pathogens such as Helicobacter pylori, Haemophilus influenzae, and Xylella fastidiosa. There are many indications that VapD proteins contribute to virulence, even though the exact mechanism is not known. VapD proteins are either encoded by stand-alone genes or form toxin-antitoxin pairs with VapX. We performed a comprehensive census of vapD-like genes and found two new antitoxins, VapW and VapY. The VapW antitoxins are catalytically inactivated variants of VapD, revealing a new evolutionary mechanism for the appearance of toxin-antitoxin pairs.

Bacillus cereus, a Gram-positive aerobic bacterium commonly found in soil, food, and water, forms endospores that can withstand harsh environmental conditions. The endospores are encased in a protective spore coat consisting of multiple layers of proteins, among which, CotE serves as a crucial morphogenetic protein within the outer coat. In this study, we observed that the homotrimeric CotE protein underwent further oligomerization induced by Ca²⁺ and was subsequently dissociated by dipicolinic acid, a compound released from the spore core during germination. Through cryo-electron microscopy and tomography analyses of the Ca²⁺-induced CotE oligomer, combined with structural predictions and biochemical studies, we propose a three-dimensional meshwork organization facilitated by tryptophan-based interactions between CotE trimers. The resulting meshwork was organized in a defective diamond-like tetrahedral configuration. These insights enhance our understanding of how CotE contributes to endospore morphogenesis and germination through the rapid disassembly of these layers.

Importance: Bacterial endospores are highly resilient structures that allow bacteria to survive extreme environmental conditions, making them a significant concern in food safety and healthcare. The protein CotE plays a critical role in forming the protective outer coat of these endospores. Our research uncovers the three-dimensional meshwork architecture of CotE and reveals how it contributes to the structural integrity and rapid disassembly of endospores during germination. By understanding CotE's unique 3D structure and its interaction with other molecules, we gain valuable insights into how bacterial endospores are formed and how they can be effectively targeted for sterilization. This work not only advances our fundamental knowledge of bacterial endospore biology but also has potential applications in developing new strategies to combat bacterial contamination and improve sterilization techniques in the food and healthcare industries.

Pseudomonas aeruginosa, a Gram-negative pathogen, has emerged as one of the most highly antibiotic-resistant bacteria worldwide and subsequently has become a leading cause of healthcare-associated, life-threatening infections. P. aeruginosa multidrug efflux Y (MexY) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. It is a major determinant for resistance to aminoglycosides in this opportunistic pathogen. However, the detailed molecular mechanisms involved in aminoglycoside recognition and extrusion by MexY have not been elucidated. Here, we report the cryo-electron microscopy structure of MexY to a resolution of 3.63 Å. The structure directly indicates two plausible pathways for drug export. It also suggests that MexY is capable of picking up antibiotics via the ceiling of the central cavity formed by the MexY trimer. Molecular dynamics simulations depict that MexY is able to use a tunnel connecting the central cavity to the funnel of the trimer to export its substrates.

Importance: Here, we report the cryo-electron microscopy structure of the MexY multidrug efflux pump, posing the possibility that this pump is capable of capturing antibiotics from both the central cavity and the periplasmic cleft of the pump. The results indicate that MexY may utilize charged residues to bind and export drugs, mediating resistance to these antibiotics.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in CFTR protein dysfunction. CFTR dysfunction has multi-organ consequences, leading to dehydrated mucus that is adherent to epithelia. In the lungs, this leads to recalcitrant infections with bacteria such as Pseudomonas aeruginosa. In the gut, mucus-laden feces can adhere to the intestines, resulting in distal intestinal obstruction syndrome (DIOS). There is limited information on how lung colonization and DIOS are correlated in people with CF (pwCF). In this novel work, we describe the development of spontaneous lung colonization of CF pathogens in young (<3 months old) CF rats, preceding the development of DIOS. Once DIOS is established, the lung microbiome becomes predominated by taxa also observed in the feces. Induced infection with P. aeruginosa in the CF rats reflects data found in pwCF, as once CF rats are infected, they retain a higher relative abundance of P. aeruginosa than their healthy agemates. Finally, we found that ivacaftor treatment favors a healthier gut microbiome in CF rats, decreasing the relative abundance of Escherichia coli. These results indicate that the CF rat model is recapitulative of human CF disease with the spontaneous lung colonization of traditional CF pathogens and maintenance of P. aeruginosa after induced infection. Furthermore, these results indicate a possible role for the gut-lung axis in lung colonization and DIOS in CF.IMPORTANCEThese data describe for the first time the development of spontaneous lung colonization in the cystic fibrosis (CF) rat model, a hallmark aspect of human CF disease. We also find that CF rats infected with Pseudomonas aeruginosa maintain higher relative abundance following chronic infection as compared to healthy rats, similar to those is seen in people with CF. Additionally, we describe the possible contribution of the gut-lung axis linking lung health with distal intestinal obstruction syndrome, a relationship largely unexplored in the context of CF.

Analyses of codon usage in eukaryotes suggest that amino acid usage responds to GC pressure so AT-biased substitutions drive higher usage of amino acids with AT-ending codons. Here, we combine single-cell transcriptomics and phylogenomics to explore codon usage patterns in foraminifera, a diverse and ancient clade of predominantly uncultivable microeukaryotes. We curate data from 1,044 gene families in 49 individuals representing 28 genera, generating perhaps the largest existing dataset of data from a predominantly uncultivable clade of protists, to analyze compositional bias and codon usage. We find extreme variation in composition, with a median GC content at fourfold degenerate silent sites below 3% in some species and above 75% in others. The most AT-biased species are distributed among diverse non-monophyletic lineages. Surprisingly, despite the extreme variation in compositional bias, amino acid usage is highly conserved across all foraminifera. By analyzing nucleotide, codon, and amino acid composition within this diverse clade of amoeboid eukaryotes, we expand our knowledge of patterns of genome evolution across the eukaryotic tree of life.IMPORTANCEPatterns of molecular evolution in protein-coding genes reflect trade-offs between substitution biases and selection on both codon and amino acid usage. Most analyses of these factors in microbial eukaryotes focus on model species such as Acanthamoeba, Plasmodium, and yeast, where substitution bias is a primary contributor to patterns of amino acid usage. Foraminifera, an ancient clade of single-celled eukaryotes, present a conundrum, as we find highly conserved amino acid usage underlain by divergent nucleotide composition, including extreme AT-bias at silent sites among multiple non-sister lineages. We speculate that these paradoxical patterns are enabled by the dynamic genome structure of foraminifera, whose life cycles can include genome endoreplication and chromatin extrusion.

The protozoan parasite Plasmodium falciparum, which is responsible for the deadliest form of human malaria, accounts for over half a million deaths a year. These parasites proliferate in human red blood cells by consecutive rounds of closed mitoses called schizogony. Their virulence is attributed to their ability to modify the infected red cells to adhere to the vascular endothelium and to evade immunity through antigenic switches. Spatial dynamics at the nuclear periphery were associated with the regulation of processes that enable the parasites to establish long-term infection. However, our knowledge of components of the nuclear envelope (NE) in Plasmodium remains limited. One of the major protein complexes at the NE is the linker of nucleoskeleton and cytoskeleton (LINC) complex that forms a connecting bridge between the cytoplasm and the nucleus through the interaction of SUN and KASH domain proteins. Here, we have identified two SUN-domain proteins as possible components of the LINC complex of P. falciparum and show that their proper expression is essential for the parasite's proliferation in human red blood cells, and their depletion leads to the formation of membranous whorls and morphological changes of the NE. In addition, their differential expression highlights different functions at the nuclear periphery as PfSUN2 is specifically associated with heterochromatin, while PfSUN1 expression is essential for activation of the DNA damage response. Our data provide indications for the involvement of the LINC complex in crucial biological processes in the intraerythrocytic development cycle of malaria parasites.

Importance: Plasmodium falciparum, the parasite causing the deadliest form of malaria, is able to thrive in its human host by tight regulation of cellular processes, orchestrating nuclear dynamics with cytoplasmic machineries that are separated by the nuclear envelope. One of the major protein complexes that connect nuclear and cytoplasmic processes in eukaryotes is the linker of nucleoskeleton and cytoskeleton (LINC) complex. However, while the nuclear periphery of P. falciparum was implicated in several important functions, the role of the LINC complex in Plasmodium biology is unknown. Here, we identify two components of P. falciparum LINC complex and demonstrate that they are essential for the parasites' proliferation in human blood, and their depletion leads to the formation of morphological changes in the cell. In addition, the two components have different functions in activating the DNA damage response and in their association with heterochromatin. Our data provide evidence for their essential roles in the parasites' cell cycle.