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Microbiology Spectrum Jun 2024We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of . We report the discovery of monoclonal antibodies...
We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of . We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy reported in that model. Our work shows that expression of type 3 fimbriae in culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of infection (different sites in the body) as well as against biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of . We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling in biofilms and during infection.
PubMed: 38940542
DOI: 10.1128/spectrum.00400-24 -
Frontiers in Cellular and Infection... 2024Enteroaggregative (EAEC) is a major cause of diarrhea worldwide. EAEC are highly adherent to cultured epithelial cells and make biofilms. Both adherence and biofilm...
Enteroaggregative (EAEC) is a major cause of diarrhea worldwide. EAEC are highly adherent to cultured epithelial cells and make biofilms. Both adherence and biofilm formation rely on the presence of aggregative adherence fimbriae (AAF). We compared biofilm formation from two EAEC strains of each of the five AAF types. We found that AAF type did not correlate with the level of biofilm produced. Because the composition of the EAEC biofilm has not been fully described, we stained EAEC biofilms to determine if they contained protein, carbohydrate glycoproteins, and/or eDNA and found that EAEC biofilms contained all three extracellular components. Next, we assessed the changes to the growing or mature EAEC biofilm mediated by treatment with proteinase K, DNase, or a carbohydrate cleavage agent to target the different components of the matrix. Growing biofilms treated with proteinase K had decreased biofilm staining for more than half of the strains tested. In contrast, although sodium metaperiodate only altered the biofilm in a quantitative way for two strains, images of biofilms treated with sodium metaperiodate showed that the EAEC were more spread out. Overall, we found variability in the response of the EAEC strains to the treatments, with no one treatment producing a biofilm change for all strains. Finally, once formed, mature EAEC biofilms were more resistant to treatment than biofilms grown in the presence of those same treatments.
Topics: Biofilms; Endopeptidase K; Escherichia coli; Deoxyribonucleases; Fimbriae, Bacterial; Bacterial Adhesion; Humans; Periodic Acid
PubMed: 38938878
DOI: 10.3389/fcimb.2024.1379206 -
Microbiology Spectrum Jun 2024O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from and induces a massive...
UNLABELLED
O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that O1 isolated from stool is genetically similar to recovered from the upper intestinal tract.
IMPORTANCE
O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed genomes from stool are usually studied, but isolated from vomit may be more representative of where colonizes in the upper intestinal epithelium. may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in genetic diversity, and only in some patients. These results fill a gap in our understanding of the life cycle, transmission, and evolution.
PubMed: 38916318
DOI: 10.1128/spectrum.00785-24 -
MSphere Jun 2024Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a...
Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a critical step required for any pathogen to establish infection by anchoring a bacterium to host tissue. The human pathogen enterohemorrhagic (EHEC) O157:H7encodes 16 fimbriae that may be important for EHEC to initiate infection and allow for productive expression of virulence traits important in later stages of infection, including a type III secretion system (T3SS) and Shiga toxin; however, the roles of most EHEC fimbriae are largely uncharacterized. Here, we provide evidence that two EHEC fimbriae, Yad and Yeh, modulate expression of diverse genes including genes encoding T3SS and Shiga toxin and that these fimbriae are required for robust colonization of the gastrointestinal tract. These findings reveal a significant and previously unappreciated role for fimbriae in bacterial pathogenesis as important determinants of virulence gene expression.IMPORTANCEFimbriae are extracellular proteinaceous structures whose defining role is to anchor bacteria to surfaces. This is a fundamental step for bacterial pathogens to establish infection in a host. Here, we show that the contributions of fimbriae to pathogenesis are more complex. Specifically, we demonstrate that fimbriae influence expression of virulence traits essential for disease progression in the intestinal pathogen enterohemorrhagic . Gram-positive and Gram-negative bacteria express multiple fimbriae; therefore, these findings may have broad implications for understanding how pathogens use fimbriae, beyond adhesion, to initiate infection and coordinate gene expression, which ultimately results in disease.
PubMed: 38904402
DOI: 10.1128/msphere.00124-24 -
Infection and Drug Resistance 2024The most common extraintestinal pathogen and infection site is uropathogenic (UPEC), which causes urinary tract infections (UTIs). UPEC is also a common pathogen in...
OBJECTIVE
The most common extraintestinal pathogen and infection site is uropathogenic (UPEC), which causes urinary tract infections (UTIs). UPEC is also a common pathogen in bloodstream infections; in severe cases, it can lead to death. Although host and bacterial virulence factors have been demonstrated to be associated with UTI pathogenesis, the role of the related contributing factors in UTI and urinary source bacteremia is not yet fully understood. This study aimed to compare and analyze the factors contributing to urinary bacteremia in patients with UTI.
METHODS
A total of 171 strains collected from patients with UTI and urinary source bacteremia at Chiayi Christian Hospital were used. Phylogenetic groups and virulence factors were determined using PCR. Drug resistance patterns were determined using the disk diffusion assay.
RESULTS
Previous studies have demonstrated that fimbriae and papGII may be associated with first-step infections and severe UTIs, respectively. As expected, highly virulent strains (belonging to the phylogenetic B2 and D groups) were dominant in the bacteremic UTI (90%) and UTI (86.27%) groups. However, our results showed that the UTI group had a significantly higher prevalence of (belonging to the S and FIC fimbriae) than the bacteremic UTI group (29.4% vs 12.5%; p=0.008). In the bacteremic group, we found that / was only detected in highly virulent strains. The bacteremic UTI group had a significantly higher prevalence of GII (belonging to P fimbriae) than the UTI group (55.8% vs 37.3%; p=0.026). In addition, the P fimbriae gene cluster, including , and , was predominant in highly virulent strains. Notably, our results show that multidrug-resistant (MDR) strains were significantly less virulent than non MDR strains.
CONCLUSION
Taken together, our results provide insights into the contributing factors in patients with UTI and urinary bacteremia.
PubMed: 38903152
DOI: 10.2147/IDR.S458925 -
Applied and Environmental Microbiology Jun 2024Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a...
UNLABELLED
Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures ("py"') demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various - and -type cytochromes was observed in "py," pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells' photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(-), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis.
IMPORTANCE
Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet's history.
PubMed: 38899885
DOI: 10.1128/aem.00863-24 -
Virulence Dec 2024The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms....
The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low Amp and High Amp, respectively. Whole-genome sequencing revealed that both Low and High Amp strains contained mutations in the , , and genes. The High Amp strain exhibited a single additional mutation in the gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the Amp strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the Amp strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment , implicating the increasing difficulty in treating host infections caused by the Amp strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.
Topics: Uropathogenic Escherichia coli; Animals; Drug Resistance, Multiple, Bacterial; Urinary Tract Infections; Escherichia coli Infections; Mice; Anti-Bacterial Agents; Ampicillin; Mutation; Escherichia coli Proteins; Female; Humans; Microbial Sensitivity Tests; Whole Genome Sequencing
PubMed: 38899601
DOI: 10.1080/21505594.2024.2367648 -
BMC Genomics Jun 2024Adhesins are crucial factors in the virulence of bacterial pathogens such as Escherichia coli. However, to date no resources have been dedicated to the detailed analysis...
Adhesins are crucial factors in the virulence of bacterial pathogens such as Escherichia coli. However, to date no resources have been dedicated to the detailed analysis of E. coli adhesins. Here, we provide adhesiomeR software that enables characterization of the complete adhesin repertoire, termed the adhesiome. AdhesiomeR incorporates the most comprehensive database of E. coli adhesins and facilitates an extensive analysis of adhesiome. We demonstrate that adhesiomeR achieves 98% accuracy when compared with experimental analyses. Based on analysis of 15,000 E. coli genomes, we define novel adhesiome profiles and clusters, providing a nomenclature for a unified comparison of E. coli adhesiomes.
Topics: Adhesins, Escherichia coli; Escherichia coli; Software; Genome, Bacterial; Computational Biology
PubMed: 38886681
DOI: 10.1186/s12864-024-10525-6 -
The EMBO Journal Jun 2024Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a...
Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.
PubMed: 38886579
DOI: 10.1038/s44318-024-00135-z -
Nature Communications Jun 2024Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns...
Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers.
Topics: Cryoelectron Microscopy; Fimbriae Proteins; Archaeal Proteins; Models, Molecular; Fimbriae, Bacterial; Protein Conformation; Amino Acid Sequence
PubMed: 38877064
DOI: 10.1038/s41467-024-45062-z