The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin.

BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

The strongest protein binder is surprisingly labile.

Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Crystal structure of the long Rib domain of the LPXTG-anchored surface protein from Limosilactobacillus reuteri.

The Rib domain, which is often found as tandem-repeat structural modules in surface proteins of Gram-positive bacteria, plays important roles in mediating interactions of bacteria with their environments and hosts. A comprehensive structural analysis of various Rib domains is essential to fully understand their impact on the structure and functionality of these bacterial adhesins. To date, structural information has been limited for this expansive group of domains. In this study, the high-resolution crystal structure of the second member of the long Rib domain, a unique subclass within the Rib-domain family, derived from Limosilactobacillus reuteri is presented. The data not only demonstrate a highly conserved structure within the long Rib domain, but also highlight an evolutionary convergence in structural architecture with other modular domains found in cell-adhesion molecules.

Two remarkable serine/leucine polymorphisms in Helicobacter pylori: functional importance for serine protease HtrA and adhesin BabA.

Single nucleotide polymorphisms (SNPs) account for significant genomic variability in microbes, including the highly diverse gastric pathogen Helicobacter pylori. However, data on the effects of specific SNPs in pathogen-host interactions are scarce. Recent functional studies unravelled how a serine/leucine polymorphism in serine protease HtrA affects the formation of proteolytically active trimers and modulates cleavage of host cell-to-cell junction proteins during infection. A similar serine/leucine mutation in the carbohydrate binding domain of the adhesin BabA controls binding of ABO blood group antigens, enabling binding of either only the short Lewis b/H antigens of blood group O or also the larger antigens of blood groups A and B. Here we summarize the functional importance of these two remarkable bacterial SNPs and their effect on the outcome of pathogen-host interactions.

Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

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.

Molecular insights into the fine-tuning of pH-dependent ArsR-mediated regulation of the SabA adhesin in Helicobacter pylori.

Adaptation to variations in pH is crucial for the ability of Helicobacter pylori to persist in the human stomach. The acid responsive two-component system ArsRS, constitutes the global regulon that responds to acidic conditions, but molecular details of how transcription is affected by the ArsR response regulator remains poorly understood. Using a combination of DNA-binding studies, in vitro transcription assays, and H. pylori mutants, we demonstrate that phosphorylated ArsR (ArsR-P) forms an active protein complex that binds DNA with high specificity in order to affect transcription. Our data showed that DNA topology is key for DNA binding. We found that AT-rich DNA sequences direct ArsR-P to specific sites and that DNA-bending proteins are important for the effect of ArsR-P on transcription regulation. The repression of sabA transcription is mediated by ArsR-P with the support of Hup and is affected by simple sequence repeats located upstream of the sabA promoter. Here stochastic events clearly contribute to the fine-tuning of pH-dependent gene regulation. Our results reveal important molecular aspects for how ArsR-P acts to repress transcription in response to acidic conditions. Such transcriptional control likely mediates shifts in bacterial positioning in the gastric mucus layer.

Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

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.

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.

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.

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.

Gingipains may be one of the key virulence factors of Porphyromonas gingivalis to impair cognition and enhance blood-brain barrier permeability: An animal study.

AIM: Blood-brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process. MATERIALS AND METHODS: C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav-1 levels were measured. The mechanisms of Ddx3x-induced regulation on Mfsd2a by arginine-specific gingipain A (RgpA) in BMECs were explored. RESULTS: P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up-regulated Cav-1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav-1 regulatory axis was activated. CONCLUSIONS: Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav-1 axis.

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.

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.

Porphyromonas gingivalis and the pathogenesis of Alzheimer's disease.

The cause of Alzheimer's disease (AD), and the pathophysiological mechanisms involved, remain major unanswered questions in medical science. Oral bacteria, especially those species associated with chronic periodontitis and particularly Porphyromonas gingivalis, are being linked causally to AD pathophysiology in a subpopulation of susceptible individuals. P. gingivalis produces large amounts of proteolytic enzymes, haem and iron capture proteins, adhesins and internalins that are secreted and attached to the cell surface and concentrated onto outer membrane vesicles (OMVs). These enzymes and adhesive proteins have been shown to cause host tissue damage and stimulate inflammatory responses. The ecological and pathophysiological roles of P. gingivalis OMVs, their ability to disperse widely throughout the host and deliver functional proteins lead to the proposal that they may be the link between a P. gingivalis focal infection in the subgingivae during periodontitis and neurodegeneration in AD. P. gingivalis OMVs can cross the blood brain barrier and may accelerate AD-specific neuropathology by increasing neuroinflammation, plaque/tangle formation and dysregulation of iron homeostasis, thereby inducing ferroptosis leading to neuronal death and neurodegeneration.

Serine-rich repeat proteins: well-known yet little-understood bacterial adhesins.

Serine-rich-repeat proteins (SRRPs) are large mucin-like glycoprotein adhesins expressed by a plethora of pathogenic and symbiotic Gram-positive bacteria. SRRPs play major functional roles in bacterial-host interactions, like adhesion, aggregation, biofilm formation, virulence, and pathogenesis. Through their functional roles, SRRPs aid in the development of host microbiomes but also diseases like infective endocarditis, otitis media, meningitis, and pneumonia. SRRPs comprise shared domains across different species, including two or more heavily O-glycosylated long stretches of serine-rich repeat regions. With loci that can be as large as ~40 kb and can encode up to 10 distinct glycosyltransferases that specifically facilitate SRRP glycosylation, the SRRP loci makes up a significant portion of the bacterial genome. The significance of SRRPs and their glycans in host-microbe communications is becoming increasingly evident. Studies are beginning to reveal the glycosylation pathways and mature O-glycans presented by SRRPs. Here we review the glycosylation machinery of SRRPs across species and discuss the functional roles and clinical manifestations of SRRP glycosylation.