Vaginal microbiomes show ethnic evolutionary dynamics and positive selection of Lactobacillus adhesins driven by a long-term niche-specific process.

The vaginal microbiome's composition varies among ethnicities. However, the evolutionary landscape of the vaginal microbiome in the multi-ethnic context remains understudied. We perform a systematic evolutionary analysis of 351 vaginal microbiome samples from 35 multi-ethnic pregnant women, in addition to two validation cohorts, totaling 462 samples from 90 women. Microbiome alpha diversity and community state dynamics show strong ethnic signatures. Lactobacillaceae have a higher ratio of non-synonymous to synonymous polymorphism and lower nucleotide diversity than non-Lactobacillaceae in all ethnicities, with a large repertoire of positively selected genes, including the mucin-binding and cell wall anchor genes. These evolutionary dynamics are driven by the long-term evolutionary process unique to the human vaginal niche. Finally, we propose an evolutionary model reflecting the environmental niches of microbes. Our study reveals the extensive ethnic signatures in vaginal microbial ecology and evolution, highlighting the importance of studying the host-microbiome ecosystem from an evolutionary perspective.

It takes two to attach - endo-1,3-ß-d-glucanase as a potential receptor of mannose-independent, FimH-dependent Salmonella Typhimurium binding to spinach leaves.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Prey killing without invasion by Bdellovibrio bacteriovorus defective for a MIDAS-family adhesin.

The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall, forming a rounded killed prey, or bdelloplast, containing a live B. bacteriovorus. Redundancy in adhesive processes makes invasive mutants rare. Here, we identify a MIDAS adhesin family protein, Bd0875, that is expressed at the predator-prey invasive junction and is important for successful invasion of prey. A mutant strain lacking bd0875 is still able to form round, dead bdelloplasts; however, 10% of the bdelloplasts do not contain B. bacteriovorus, indicative of an invasion defect. Bd0875 activity requires the conserved MIDAS motif, which is linked to catch-and-release activity of MIDAS proteins in other organisms. A proteomic analysis shows that the uninvaded bdelloplasts contain B. bacteriovorus proteins, which are likely secreted into the prey by the Δbd0875 predator during an abortive invasion period. Thus, secretion of proteins into the prey seems to be sufficient for prey killing, even in the absence of a live predator inside the prey periplasm.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Unravelling the Antimicrobial, Antibiofilm, Suppressing Fibronectin Binding Protein A (fnba) and cna Virulence Genes, Anti-Inflammatory and Antioxidant Potential of Biosynthesized Solanum lycopersicum Silver Nanoparticles.

Background and Objectives: Urinary tract infections [UTIs] are considered the third most known risk of infection in human health around the world. There is increasing appreciation for the pathogenicity of Gram-positive and Gram-negative strains in UTIs, aside from fungal infection, as they have numerous virulence factors. Materials and Methods: In this study, fifty urine samples were collected from patients suffering from UTI. Among the isolates of UTI microbes, six isolates were described as MDR isolates after an antibiotic susceptibility test carried out using ten different antibiotics. An alternative treatment for microbial elimination involved the use of biosynthesized silver nanoparticles (AgNPs) derived from Solanum lycopersicum [S. cumin]. Results: The sizes and shapes of AgNPs were characterized through TEM imaging, which showed spherical particles in a size range of 35-80 nm, of which the average size was 53 nm. Additionally, the silver nanoparticles (AgNPs) demonstrated inhibitory activity against Staphylococcus aureus (OR648079), exhibiting a 31 mm zone of inhibition at a minimum inhibitory concentration (MIC) of 4 mg/mL and a minimum bactericidal concentration (MBC) of 8 mg/mL. This was followed by Aspergillus niger (OR648075), which showed a 30 mm inhibition zone at an MIC of 16 mg/mL and a minimum fungicidal concentration (MFC) of 32 mg/mL. Then, Enterococcus faecalis (OR648078), Klebsiella pneumoniae (OR648081), and Acinetobacter baumannii (OR648080) each displayed a 29 mm zone of inhibition at an MIC of 8 mg/mL and an MBC of 16 mg/mL. The least inhibition was observed against Candida auris (OR648076), with a 25 mm inhibition zone at an MIC of 16 mg/mL and an MFC of 32 mg/mL. Furthermore, AgNPs at different concentrations removed DPPH and H2O2 at an IC50 value of 13.54 µg/mL. Also, AgNPs at 3 mg/mL showed remarkable DNA fragmentation in all bacterial strains except Enterococcus faecalis. The phytochemical analysis showed the presence of different active organic components in the plant extract, which concluded that rutin was 88.3 mg/g, garlic acid was 70.4 mg/g, and tannic acid was 23.7 mg/g. Finally, AgNPs concentrations in the range of 3-6 mg/mL showed decreased expression of two of the fundamental genes necessary for biofilm formation within Staphylococcus aureus, fnbA (6 folds), and Cna (12.5 folds) when compared with the RecA gene, which decreased by one-fold when compared with the control sample. These two genes were submitted with NCBI accession numbers [OR682119] and [OR682118], respectively. Conclusions: The findings from this study indicate that biosynthesized AgNPs from Solanum lycopersicum exhibit promising antimicrobial and antioxidant properties against UTI pathogens, including strains resistant to multiple antibiotics. This suggests their potential as an effective alternative treatment for UTIs. Further research is warranted to fully understand the mechanisms of action and to explore the therapeutic applications of these nanoparticles in combating UTIs.

The spontaneously produced lysogenic prophage phi456 promotes bacterial resistance to adverse environments and enhances the colonization ability of avian pathogenic Escherichia coli strain DE456.

In the last decade, prophages that possess the ability of lysogenic transformation have become increasingly significant. Their transfer and subsequent activity in the host have a significant impact on the evolution of bacteria. Here, we investigate the role of prophage phi456 with high spontaneous induction in the bacterial genome of Avian pathogenic Escherichia coli (APEC) DE456. The phage particles, phi456, that were released from DE456 were isolated, purified, and sequenced. Additionally, phage particles were no longer observed either during normal growth or induced by nalidixic acid in DE456Δphi456. This indicated that the released phage particles from DE456 were only phi456. We demonstrated that phi456 contributed to biofilm formation through spontaneous induction of the accompanying increase in the eDNA content. The survival ability of DE456Δphi456 was decreased in avian macrophage HD11 under oxidative stress and acidic conditions. This is likely due to a decrease in the transcription levels of three crucial genes-rpoS, katE, and oxyR-which are needed to help the bacteria adapt to and survive in adverse environments. It has been observed through animal experiments that the presence of phi456 in the DE456 genome enhances colonization ability in vivo. Additionally, the number of type I fimbriae in DE456Δphi456 was observed to be reduced under transmission electron microscopy when compared to the wild-type strain. The qRT-PCR results indicated that the expression levels of the subunit of I fimbriae (fimA) and its apical adhesin (fimH) were significantly lower in DE456Δphi456. Therefore, it can be concluded that phi456 plays a crucial role in helping bacterial hosts survive in unfavorable conditions and enhancing the colonization ability in DE456.

Candida auris undergoes adhesin-dependent and -independent cellular aggregation.

Candida auris is a fungal pathogen of humans responsible for nosocomial infections with high mortality rates. High levels of resistance to antifungal drugs and environmental persistence mean these infections are difficult to treat and eradicate from a healthcare setting. Understanding the life cycle and the genetics of this fungus underpinning clinically relevant traits, such as antifungal resistance and virulence, is of the utmost importance to develop novel treatments and therapies. Epidemiological and genomic studies have identified five geographical clades (I-V), which display phenotypic and genomic differences. Aggregation of cells, a phenotype primarily of clade III strains, has been linked to reduced virulence in some infection models. The aggregation phenotype has thus been associated with conferring an advantage for (skin) colonisation rather than for systemic infection. However, strains with different clade affiliations were compared to infer the effects of different morphologies on virulence. This makes it difficult to distinguish morphology-dependent causes from clade-specific or even strain-specific genetic factors. Here, we identify two different types of aggregation: one induced by antifungal treatment which is a result of a cell separation defect; and a second which is controlled by growth conditions and only occurs in strains with the ability to aggregate. The latter aggregation type depends on an ALS-family adhesin which is differentially expressed during aggregation in an aggregative C. auris strain. Finally, we demonstrate that macrophages cannot clear aggregates, suggesting that aggregation might after all provide a benefit during systemic infection and could facilitate long-term persistence in the host.

Complete genome sequence of Psychrobacter cibarius AOSW16051, a trimeric autotransporter adhesin synthesizing bacterium isolated from the Baltic Sea.

Bacteria of the genus Psychrobacter are widely distributed in the global low-temperature marine environment and have been studied for their effects on the settlement and metamorphosis of marine invertebrates. Psychrobacter cibarius AOSW16051 was isolated from the surface water samples of the Baltic Sea on the edge of the Arctic Ocean. Here, we present the complete genome of strain AOSW16051, which consists of a circular chromosome composed of 3,425,040 nucleotides with 42.98% G + C content and a circular plasmid composed of 5846 nucleotides with 38.66% G + C content. The genes predicted in this strain showed its strong outer membrane system, type VI secretion system and adhesion system. Trimeric autotransporter adhesins (TAAs) has been identified in the genome of P. cibarius AOSW16051, which has a variety of biological functions in interacting with host cells. However, there are no reports on TAAs in marine bacteria and aquatic pathogenic bacteria. By analyzing the genomic data, we can gain valuable insights to enhance our understanding of the physiological characteristics of P. cibarius, as well as the biological functions of TAAs and their role in triggering metamorphosis of invertebrate larvae.

Reconstitution of a biofilm adhesin system from a sulfate-reducing bacterium in Pseudomonas fluorescens.

Biofilms of sulfate-reducing bacterium (SRB) like Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses. Although the mechanisms of biofilm formation by DvH are not yet well understood, recent studies indicate the large adhesin, DvhA, is a key determinant of biofilm formation. The dvhA gene neighborhood resembles the biofilm-regulating Lap system of Pseudomonas fluorescens but is curiously missing the c-di-GMP-binding regulator LapD. Instead, DvH encodes an evolutionarily unrelated c-di-GMP-binding protein (DVU1020) that we hypothesized is functionally analogous to LapD. To study this unusual Lap system and overcome experimental limitations with the slow-growing anaerobe DvH, we reconstituted its predicted SRB Lap system in a P. fluorescens strain lacking its native Lap regulatory components (ΔlapGΔlapD). Our data support the model that DvhA is a cell surface-associated LapA-like adhesin with a N-terminal "retention module" and that DvhA is released from the cell surface upon cleavage by the LapG-like protease DvhG. Further, we demonstrate DVU1020 (named here DvhD) represents a distinct class of c-di-GMP-binding, biofilm-regulating proteins that regulates DvhG activity in response to intracellular levels of this second messenger. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.

Unraveling the crystal structure of the HpaA adhesin: insights into cell adhesion function and epitope localization of a Helicobacter pylori vaccine candidate.

Helicobacter pylori is a bacterium that exhibits strict host restriction to humans and non-human primates, and the bacterium is widely acknowledged as a significant etiological factor in the development of chronic gastritis, peptic ulcers, and gastric cancers. The pathogenic potential of this organism lies in its adeptness at colonizing the gastric mucosa, which is facilitated by a diverse repertoire of virulence factors, including adhesins that promote the attachment of the bacteria to the gastric epithelium. Among these adhesins, HpaA stands out due to its conserved nature and pivotal role in establishing H. pylori colonization. Moreover, this lipoprotein holds promise as an antigen for the development of effective H. pylori vaccines, thus attracting considerable attention for in-depth investigations into its molecular function and identification of binding determinants. Here, we present the elucidation of the crystallographic structure of HpaA at 2.9 Å resolution. The folding adopts an elongated protein shape, which is distinctive to the Helicobacteraceae family, and features an apical domain extension that plays a critical role in the cell-adhesion activity on gastric epithelial cells. Our study also demonstrates the ability of HpaA to induce TNF-α expression in macrophages, highlighting a novel role as an immunoregulatory effector promoting the pro-inflammatory response in vitro. These findings not only contribute to a deeper comprehension of the multifaceted role of HpaA in H. pylori pathogenesis but also establish a fundamental basis for the design and development of structure-based derivatives, aimed at enhancing the efficacy of H. pylori vaccines. IMPORTANCE: Helicobacter pylori is a bacterium that can cause chronic gastritis, peptic ulcers, and gastric cancers. The bacterium adheres to the lining of the stomach using proteins called adhesins. One of these proteins, HpaA, is particularly important for H. pylori colonization and is considered a promising vaccine candidate against H. pylori infections. In this work, we determined the atomic structure of HpaA, identifying a characteristic protein fold to the Helicobacter family and delineating specific amino acids that are crucial to support the attachment to the gastric cells. Additionally, we discovered that HpaA can trigger the production of TNF-α, a proinflammatory molecule, in macrophages. These findings provide valuable insights into how H. pylori causes disease and suggest that HpaA has a dual role in both attachment and immune activation. This knowledge could contribute to the development of improved vaccine strategies for preventing H. pylori infections.

Human PRH1, PRH2 susceptibility and resistance and Streptococcus mutans virulence phenotypes specify different microbial profiles in caries.

BACKGROUND: Lifestyle- and sucrose-dependent polymicrobial ecological shifts are a primary cause of caries in populations with high caries prevalence. In populations with low prevalence, PRH1, PRH2 susceptibility and resistance phenotypes may interact with the Streptococcus mutans adhesin cariogenicity phenotype to affect caries progression, but studies are lacking on how these factors affect the microbial profile of caries. METHODS: We analysed how the residency and infection profiles of S. mutans adhesin (SpaP A/B/C and Cnm/Cbm) phenotypes and commensal streptococci and lactobacilli influenced caries progression in a prospective case-referent sample of 452 Swedish adolescents with high (P4a), moderate (P6), and low (P1) caries PRH1, PRH2 phenotypes. Isolates of S. mutans from participants were analysed for adhesin expression and glycosylation and in vitro and in situ mechanisms related to caries activity. FINDINGS: Among adolescents with the resistant (P1) phenotype, infection with S. mutans high-virulence phenotypes was required for caries progression. In contrast, with highly (P4a) or moderately (P6) susceptible phenotypes, caries developed from a broader polymicrobial flora that included moderately cariogenic oral commensal streptococci and lactobacilli and S. mutans phenotypes. High virulence involved unstable residency and fluctuating SpaP ABC, B-1, or Cnm expression/glycosylation phenotypes, whereas low/moderate virulence involved SpaP A phenotypes with stable residency. Adhesin phenotypes did not display changes in individual host residency but were paired within individuals and geographic regions. INTERPRETATION: These results suggest that receptor PRH1, PRH2 susceptibility and resistance and S. mutans adhesin virulence phenotypes specify different microbial profiles in caries. FUNDING: Swedish Research Council and funding bodies listed in the acknowledgement section.

A Porphyromonas gingivalis hypothetical protein controlled by the type I-C CRISPR-Cas system is a novel adhesin important in virulence.

The ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen, uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis. Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans' most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P. gingivalis and impacts the ability of P. gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms.

An M cell-targeting recombinant L. lactis vaccine against four H. pylori adhesins.

The acidic environment and enzyme degradation lead to oral vaccines often having little immune effect. Therefore, it is an attractive strategy to study an effective and safe oral vaccine delivery system that can promote gastrointestinal mucosal immune responses and inhibit antigen degradation. Moreover, the antigens uptake by microfold cells (M cells) is the determining step in initiating efficient immune responses. Therefore, M cell-targeting is one promising approach for enhancing oral vaccine potency. In the present study, an M cell-targeting L. lactis surface display system (plSAM) was built to favor the multivalent epitope vaccine antigen (FAdE) to achieve effective gastrointestinal mucosal immunity against Helicobacter pylori. Therefore, a recombinant Lactococcus lactic acid vaccine (LL-plSAM-FAdE) was successfully prepared, and its immunological properties and protective efficacy were analyzed. The results showed that LL-plSAM-FAdE can secretively express the recombinant proteins SAM-FAdE and display the SAM-FAdE on the bacterial cell surface. More importantly, LL-plSAM-FAdE effectively promoted the phagocytosis and transport of vaccine antigen by M cells in the gastrointestinal tract of mice, and simulated high levels of cellular and humoral immune responses against four key H. pylori adhesins (Urease, CagL, HpaA, and Lpp20) in the gastrointestinal tract, thus enabling effective prevention of H. pylori infection and to some extent eliminating H. pylori already present in the gastrointestinal tract. KEY POINTS: • M-cell-targeting L. lactis surface display system LL- plSAM was designed • This system displays H. pylori vaccine-promoted phagocytosis and transport of M cell • A promising vaccine candidate for controlling H. pylori infection was verified.

rgpA-Engineered/Functionalized DNA Vaccine as a Novel Prophylactic Vaccination to Prevent Porphyromonas gingivalis-Induced Periodontitis: An in Vivo Study.

BACKGROUND: Arg-gingipain A (rgpA) and Arg-gingipain B (rgpB) are crucial virulence factors associated with Porphyromonas gingivalis (P. gingivalis) and have been recognized as promising targets for antibacterial vaccines. Although vaccines containing rgpA have shown efficacy, the incorporation of rgpB, which lacks the haemagglutinin adhesin (HA) domain, diminishes the vaccine's effectiveness. This study aims to assess the immunogenicity of the functional HA domain of rgpA in mouse periodontitis models. METHODS: A total of 24 mice were randomly divided into four groups, each receiving different immune injections: group A received phosphate-buffered saline (PBS) as an empty control; group B received pVAX1 as a negative control (NC); group C received pVAX1-HA; and group D received pVAX1-rgpA. The mice were subjected to intramuscular injections every two weeks for a total of three administrations. Prior to each immunization, blood samples were collected for antibody detection under isoflurane anesthesia. Following the final immunization, periodontitis was induced two weeks later by using sutures soaked in a P. gingivalis solution. The mice were euthanized after an additional two-week period. To assess the safety of the procedure, major organs were examined through hematoxylin-eosin (HE) staining. Subsequently, the levels of IgG, IgG1, and IgG2a in the serum were quantified via enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of inflammatory factors in the gingiva, including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor alpha (TNF-α), was determined using quantitative real-time reverse transcript PCR (qRT-PCR). The extent of bone loss in periodontal tissues was evaluated using micro-computed tomography (micro-CT) and HE staining. RESULTS: HE staining of the organs confirmed the absence of vaccine-induced toxicity in vivo. After the second immunization, both the rgpA and HA groups displayed significantly higher specific IgG titers in comparison to the NC and PBS groups (p < 0.05). Furthermore, the rgpA and HA groups exhibited a noteworthy predominance of IgG1 antibodies after three immunization doses, while there was a noticeable reduction in IgG2a levels observed following ligation with P. gingivalis sutures, as opposed to the NC and PBS groups (p < 0.05). Additionally, both the HA and rgpA groups showed a significant decrease in the expression of inflammatory factors such as IL-6, IL-1ß, and TNF-α, as well as a reduction in bone loss around periodontitis-affected teeth, when compared to the NC and PBS groups (p < 0.05). CONCLUSIONS: The results of this study demonstrate that the rgpA-engineered/functionalized HA gene vaccine is capable of eliciting a potent prophylactic immune response against P. gingivalis-induced periodontitis, effectively serving as an immunogenic and protective agent in vivo.

Vibrio fischeri: a model for host-associated biofilm formation.

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

Mycoplasma invasion into host cells: An integrated model of infection strategy.

Mycoplasma belong to the genus Mollicutes and are notable for their small genome sizes (500-1300 kb) and limited biosynthetic capabilities. They exhibit pathogenicity by invading various cell types to survive as intracellular pathogens. Adhesion is a crucial prerequisite for successful invasion and is orchestrated by the interplay between mycoplasma surface adhesins and specific receptors on the host cell membrane. Invasion relies heavily on clathrin- and caveolae-mediated internalization, accompanied by multiple activated kinases, cytoskeletal rearrangement, and a myriad of morphological alterations, such as membrane invagination, nuclear hypertrophy and aggregation, cytoplasmic edema, and vacuolization. Once mycoplasma successfully invade host cells, they establish resilient sanctuaries in vesicles, cytoplasm, perinuclear regions, and the nucleus, wherein specific environmental conditions favor long-term survival. Although lysosomal degradation and autophagy can eliminate most invading mycoplasmas, some viable bacteria can be released into the extracellular environment via exocytosis, a crucial factor in the prolonging infection persistence. This review explores the intricate mechanisms by which mycoplasma invades host cells and perpetuates their elusive survival, with the aim of highlighting the challenge of eradicating this enigmatic bacterium.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Sugar-binding and split domain combinations in repeats-in-toxin adhesins from Vibrio cholerae and Aeromonas veronii mediate cell-surface recognition and hemolytic activities.

Many pathogenic Gram-negative bacteria use repeats-in-toxin adhesins for colonization and biofilm formation. In the cholera agent Vibrio cholerae, flagellar-regulated hemagglutinin A (FrhA) enables these functions. Using bioinformatic analysis, a sugar-binding domain was identified in FrhA adjacent to a domain of unknown function. AlphaFold2 indicated the boundaries of both domains to be slightly shorter than previously predicted and assisted in the recognition of the unknown domain as a split immunoglobulin-like fold that can assist in projecting the sugar-binding domain toward its target. The AlphaFold2-predicted structure is in excellent agreement with the molecular envelope obtained from small-angle X-ray scattering analysis of a recombinant construct spanning the sugar-binding and unknown domains. This two-domain construct was probed by glycan micro-array screening and showed binding to mammalian fucosylated glycans, some of which are characteristic erythrocyte markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct-bound l-fucose with a Kd of 21 µM. Strikingly, this recombinant protein construct bound and lysed erythrocytes in a concentration-dependent manner, and its hemolytic activity was blocked by the addition of l-fucose. A protein ortholog construct from Aeromonas veronii was also produced and showed a similar glycan-binding pattern, binding affinity, erythrocyte-binding, and hemolytic activities. As demonstrated here with Hep-2 cells, fucose-based inhibitors of this sugar-binding domain can potentially be developed to block colonization by V. cholerae and other pathogenic bacteria that share this adhesin domain.IMPORTANCEThe bacterium, Vibrio cholerae, which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l-fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.

The ways Fusobacterium nucleatum translocate to breast tissue and contribute to breast cancer development.

Breast cancer is among the most prevalent malignancies in women worldwide. Epidemiological findings suggested that periodontal diseases may be associated with breast cancer, among which Fusobacterium nucleatum is considered an important cross-participant. In this work, we comprehensively summarize the known mechanisms of how F. nucleatum translocates to, colonizes in mammary tumors, and promotes the carcinogenesis. Specifically, F. nucleatum translocates to mammary tissue through the mammary-intestinal axis, direct nipple contact, and hematogenous transmission. Subsequently, F. nucleatum takes advantage of fusobacterium autotransporter protein 2 to colonize breast cancer and uses virulence factors fusobacterium adhesin A and lipopolysaccharide to promote proliferation. Moreover, the upregulated matrix metalloproteinase-9 induced by F. nucleatum does not only trigger the inflammatory response but also facilitates the tumor-promoting microenvironment. Aside from the pro-inflammatory effect, F. nucleatum may also be engaged in tumor immune evasion, which is achieved through the action of virulence factors on immune checkpoint receptors highly expressed on T cells, natural killer cells, and tumor-infiltrating lymphocytes. Taking breast cancer as an example, more relevant research studies may expand our current knowledge of how oral microbes affect systemic health. Hopefully, exploring these mechanisms in depth could provide new strategies for safer and more effective biologic and targeted therapies targeted at breast cancer.

High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA.

Infections caused by Campylobacter spp. are a major cause of severe enteritis worldwide. Multifactorial prevention strategies are necessary to reduce the prevalence of Campylobacter. In particular, antiadhesive strategies with specific inhibitors of early host-pathogen interaction are promising approaches to reduce the bacterial load. An in vitro flow cytometric adhesion assay was established to study the influence of carbohydrates on the adhesion of C. jejuni to Caco-2 cells. Chitosans with a high degree of polymerization and low degree of acetylation were identified as potent antiadhesive compounds, exerting significant reduction of C. jejuni adhesion to Caco-2 cells at non-toxic concentrations. Antiadhesive and also anti-invasive effects were verified by confocal laser scanning microscopy. For target identification, C. jejuni adhesins FlpA and JlpA were expressed in Escherichia coli ArcticExpress, and the influence of chitosan on binding to fibronectin and HSP90α, respectively, was investigated. While no effects on FlpA binding were found, a strong inhibition of JlpA-HSP90α binding was observed. To simulate real-life conditions, chicken meat was inoculated with C. jejuni, treated with antiadhesive chitosan, and the bacterial load was quantified. A strong reduction of C. jejuni load was observed. Atomic force microscopy revealed morphological changes of C. jejuni after 2 h of chitosan treatment, indicating disturbance of the cell wall and sacculi formation by electrostatic interaction of positively charged chitosan with the negatively charged cell surface. In conclusion, our data indicate promising antiadhesive and anti-invasive potential of high molecular weight, strongly de-acetylated chitosans for reducing C. jejuni load in livestock and food production. KEY POINTS: • Antiadhesive effects of chitosan with high DP/low DA against C. jejuni to host cells • Specific targeting of JlpA/Hsp90α interaction by chitosan • Meat treatment with chitosan reduces C. jejuni load.