The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

Human Adaptive Immunity Rescues an Inborn Error of Innate Immunity.

The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.

The molecular architecture of Lactobacillus S-layer: Assembly and attachment to teichoic acids.

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer's pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.

Immunization with a peptide mimicking lipoteichoic acid induces memory B cells in BALB/c mice.

BACKGROUND: There is an urgent clinical need for developing novel immunoprophylaxis and immunotherapy strategies against Staphylococcus aureus (S. aureus). In our previous work, immunization with a tetra-branched multiple antigenic peptide, named MAP2-3 that mimics lipoteichoic acid, a cell wall component of S. aureus, successfully induced a humoral immune response and protected BALB/c mice against S. aureus systemic infection. In this study, we further investigated whether vaccination with MAP2-3 can elicit immunologic memory. METHODS: BALB/c mice were immunized with MAP2-3 five times. After one month of the last vaccination, mice were challenged with heat-killed S. aureus via intraperitoneal injection. After a 7-day inoculation, the percentage of plasma cells, memory B cells, effector memory T cells, and follicular helper T cells were detected by flow cytometry. The levels of IL-6, IL-21, IL-2, and IFN-γ were measured by real-time PCR and ELISA. Flow cytometry results were compared by using one-way ANOVA or Mann-Whitney test, real-time PCR results were compared by using one-way ANOVA, and ELISA results were compared by using one-way ANOVA or student's t-test. RESULTS: The percentage of plasma cells and memory B cells in the spleen and bone marrow from the MAP2-3 immunized mice was significantly higher than that from the control mice. The percentage of effector memory T cells in spleens and lymphoid nodes as well as follicular helper T cells in spleens from the MAP2-3 immunized mice were also higher. Moreover, the levels of IL-6 and IL-21, two critical cytokines for the development of memory B cells, were significantly higher in the isolated splenocytes from immunized mice after lipoteichoic acid stimulation. CONCLUSIONS: Immunization with MAP2-3 can efficiently induce memory B cells and memory T cells.

Lacticaseibacillus casei K11 exerts immunomodulatory effects by enhancing natural killer cell cytotoxicity via the extracellular regulated-protein kinase pathway.

PURPOSE: Probiotics can serve as immunomodulators that regulate the activation of immune cells. This study aimed to screen potential probiotic strains that can enhance NK cell toxicity to improve host immunity. METHODS: In this investigation, we examined three potential probiotic strains, namely Lactiplantibacillus plantarum YZX21 (YZX21), Bifidobacterium bifidum FL-276.1 (FL-276.1) and Lacticaseibacillus casei K11 (K11), to assess their capacity in modulating NK cytotoxicity both in vitro and in vivo, while elucidating the underlying mechanisms involved. RESULTS: The findings demonstrated that K11 exhibited superior efficacy in enhancing NK cytotoxicity. Subsequent analysis revealed that K11 significantly augmented the secretion of perforin and granzyme B by NK cells through activation of receptors NKp30 and NKp46 via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, heat-inactivated K11 also enhanced NK cell activity to an extent comparable to live bacteria, with lipoteichoic acid from K11 identified as a crucial factor mediating the activation of NK cell cytotoxicity. CONCLUSION: Our study suggests that K11 may have potential applications as probiotics or postbiotics for regulating NK cell cytotoxicity to enhance immunity.

A Novel Antimicrobial Mechanism of Azalomycin F Acting on Lipoteichoic Acid Synthase and Cell Envelope.

Lipoteichoic acid (LTA) plays an essential role in bacterial growth and resistance to antibiotics, and LTA synthetase (LtaS) was considered as an attractive target for combating Gram-positive infections. Azalomycin F, a natural guanidyl-containing polyhydroxy macrolide, can target the LTA of Staphylococcus aureus. Using various technologies including enzyme-linked immunosorbent assay, transmission electron microscope, proteomics, and parallel reaction monitoring, here, the experimental results indicated that azalomycin F can accelerate the LTA release and disrupt the cell envelope, which would also lead to the feedback upregulation on the expressions of LtaS and other related enzymes. Simultaneously, the reconstituted enzyme activity evaluations showed that azalomycin F can significantly inhibit the extracellular catalytic domain of LtaS (eLtaS), while this was vague for LtaS embedded in the liposomes. Subsequently, the fluorescence analyses for five incubation systems containing azalomycin F and eLtaS or the LtaS-embedded liposome indicated that azalomcyin F can spontaneously bind to the active center of LtaS. Combining the mass spectroscopy analyses and the molecular dockings, the results further indicated that this interaction involves the binding sites of substrates and the LTA prolongation, especially the residues Lys299, Phe353, Trp354 and His416. All these suggested that azalomycin F has multiple antibacterial mechanisms against S. aureus. It can not only inhibit LTA biosynthesis through the interactions of its guanidyl side chain with the active center of LtaS but also disrupt the cell envelope through the synergistic effect of accelerating the LTA release, damaging the cell membrane, and electrostatically interacting with LTA. Simultaneously, these antibacterial mechanisms exhibit a synergistic inhibition effect on S. aureus cells, which would eventually cause the cellular autolysis.

Lactobacillus-derived components for inhibiting biofilm formation in the food industry.

Biofilm, a microbial community formed by especially pathogenic and spoilage bacterial species, is a critical problem in the food industries. It is an important cause of continued contamination by foodborne pathogenic bacteria. Therefore, removing biofilm is the key to solving the high pollution caused by foodborne pathogenic bacteria in the food industry. Lactobacillus, a commonly recognized probiotic that is healthy for consumer, have been proven useful for isolating the potential biofilm inhibitors. However, the addition of surface components and metabolites of Lactobacillus is not a current widely adopted biofilm control strategy at present. This review focuses on the effects and preliminary mechanism of action on biofilm inhibition of Lactobacillus-derived components including lipoteichoic acid, exopolysaccharides, bacteriocins, secreted protein, organic acids and some new identified molecules. Further, the review discusses several modern biofilm identification techniques and particularly interesting new technology of biofilm inhibition molecules. These molecules exhibit stronger inhibition of biofilm formation, playing a pivotal role in food preservation and storage. Overall, this review article discusses the application of biofilm inhibitors produced by Lactobacillus, which would greatly aid efforts to eradicate undesirable bacteria from environment in the food industries.

Crucial Role for Lipoteichoic Acid Assembly in the Metabolic Versatility and Antibiotic Resistance of Staphylococcus aureus.

Staphylococcus aureus is a public health threat due to the prevalence of antibiotic resistance and the capacity of this organism to infect numerous organs in vertebrates. To generate energy needed to proliferate within tissues, S. aureus transitions between aerobic respiration and fermentation. Fermentation results in a distinct colony morphology called the small-colony variant (SCV) due to decreased membrane potential and ATP production. These traits promote increased resistance to aminoglycoside antibiotics. Consequently, SCVs are associated with persistent infections. We hypothesize that dedicated physiological pathways support fermentative growth of S. aureus that represent potential targets for treatment of resistant infections. Lipoteichoic acid (LTA) is an essential component of the Gram-positive cell envelope that functions to maintain ion homeostasis, resist osmotic stress, and regulate autolytic activity. Previous studies revealed that perturbation of LTA reduces viability of metabolically restricted S. aureus, but the mechanism by which LTA supports S. aureus metabolic versatility is unknown. Though LTA is essential, the enzyme that synthesizes the modified lipid anchor, YpfP, is dispensable. However, ypfP mutants produce altered LTA, leading to elongation of the polymer and decreased cell association. We demonstrate that viability of ypfP mutants is significantly reduced upon environmental and genetic induction of fermentation. This anaerobic viability defect correlates with decreased membrane potential and is restored upon cation supplementation. Additionally, ypfP suppressor mutants exhibiting restored anaerobic viability harbor compensatory mutations in the LTA biosynthetic pathway that restore membrane potential. Overall, these results demonstrate that LTA maintains membrane potential during fermentative proliferation and promotes S. aureus metabolic versatility.

Lipoteichoic acid inhibits osteoclast differentiation and bone resorption via interruption of gelsolin-actin dissociation.

Bone resorption can be caused by excessive differentiation and/or activation of bone-resorbing osteoclasts. While microbe-associated molecular patterns can influence the differentiation and activation of bone cells, little is known about the role of lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, in the regulation of bone metabolism. In this study, we investigated the effect of LTA on bone metabolism using wild-type Staphylococcus aureus and the LTA-deficient mutant strain. LTA-deficient S. aureus induced higher bone loss and osteoclast differentiation than wild-type S. aureus. LTA isolated from S. aureus (SaLTA) inhibited osteoclast differentiation from committed osteoclast precursors in the presence of various osteoclastogenic factors by downregulating the expression of NFATc1. Remarkably, SaLTA attenuated the osteoclast differentiation from committed osteoclast precursors of TLR2-/- or MyD88-/- mice and from the committed osteoclast precursors transfected with paired immunoglobulin-like receptor B-targeting siRNA. SaLTA directly interacted with gelsolin, interrupting the gelsolin-actin dissociation which is a critical process for osteoclastogenesis. Moreover, SaLTA suppressed the mRNA expression of dendritic cell-specific transmembrane protein, ATPase H+ transporting V0 subunit D2, and Integrin, which encode proteins involved in cell-cell fusion of osteoclasts. Notably, LTAs purified from probiotics, including Bacillus subtilis, Enterococcus faecalis, and Lactobacillus species, also suppressed Pam2CSK4- or RANKL-induced osteoclast differentiation. Taken together, these results suggest that LTAs have anti-resorptive activity through the inhibition of osteoclastogenesis by interfering with the gelsolin-actin dissociation and may be used as effective therapeutic agents for the prevention or treatment of inflammatory bone diseases.

Glucolipids and lipoteichoic acids affect the activity of SigI, an alternative sigma factor, and WalKR, an essential two-component system, in Bacillus subtilis.

In a Bacillus subtilis ugtP mutant lacking glucolipids, SigI was activated in the log phase, and the activation of SigI in the mutant was suppressed by the expression of native ugtP. By contrast, SigI was inhibited in a yfnI mutant lacking one of the lipoteichoic acid (LTA) synthase genes, and the inhibition was suppressed by the expression of yfnI. A series of mutation analyses of the sigI promoter revealed that the two WalR binding sites were involved in the increase of PsigI -lacZ activity in the ugtP mutant and decrease of the lacZ activity in the yfnI mutant. Transcription from the SigI recognition sequence was enhanced in the ugtP mutant, whereas yfnI disruption inhibited the transcription from the SigA recognition sequence in the sigI promoter. We found that not only SigI but also WalKR, the essential two-component system, was activated in the ugtP mutant and inhibited in the yfnI mutant. The walK mutants with activated WalR exhibited abnormal morphology, but this phenotype was suppressed by the addition of MgSO4 . We conclude that glucolipids and LTA are key compounds in the maintenance of normal cell surface structure in B. subtilis.

The role of immune cells in the liver tumor microenvironment: an involvement of gut microbiota-derived factors.

More than 500 species of microbiota reside in the human intestine and coexist with humans, their host. Gut microbial metabolites and components are absorbed from the intestine and influence cells in the liver, including hepatocytes and stromal cells, such as liver sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, natural killer (NK) cells, NK T cells and other immune cells. This gut-originated axis to the liver is called the "gut-liver axis", which underscores the importance of the link between the gut and the liver. In this review, we discuss the gut microbial components and metabolites that affect cells in the liver, particularly in association with immune cells, and the related responses. We also highlight the mechanisms underlying gut microbiota-mediated liver carcinogenesis and discuss cancer prevention, including the recently clarified modulation of immune checkpoint inhibitor efficacy by the gut microbiota.

Early effects of lipoteichoic acid from Staphylococcus aureus on milk production-related signaling pathways in mouse mammary epithelial cells.

Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased ß-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal-regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus.

Lipoteichoic acid polymer length is determined by competition between free starter units.

Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (Glc2DAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstituted Staphylococcus aureus LtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred Glc2DAG starter unit, yet the use of Glc2DAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.

TLR4, MyD88, and TNF-α Expression in the Lungs of Akkaraman and Romanov Lambs in Response to LPS and LTA.

Toll-like receptors are involved in the recognition of bacterial toxins, which cause infection in the respiratory system. This study aimed to evaluate microanatomical and histological alterations in the lungs of 24 healthy Akkaraman and Romanov lambs after the administration of lipoteichoic acid (LTA), lipopolysaccharide (LPS), and LTA + LPS and investigate the gene, protein, and immune expression levels of TLR4, MyD88, and TNF-α molecules, known to have immune functions. Microanatomical examinations showed thickened peribronchial and alveolar walls in the lungs of groups LTA, LPS, and LTA + LPS of both breeds due to immune cell infiltration. TLR4, MyD88, and TNF-α immunoexpressions were positive to varying degrees in the cytoplasm and nucleus of the bronchial and bronchiolar luminal epithelial cells, alveolar epithelial cells, and alveolar macrophages. TLR4 and TNF-α protein expressions were statistically different in the LPS-treated Romanov lambs, compared to the other groups. Among the Akkaraman lambs, TLR4 gene expression was significantly higher in group LPS, and among the Romanov lambs, TLR4, MyD88, and TNF-α gene expressions were significantly higher in group LTA + LPS. Therefore, TLR4, MyD88, and TNF-α molecules, involved in the immune response, were found to be expressed at different levels against LTA and LPS in the lungs of two different sheep breeds.

Effect of forsythoside A on the transcriptional profile of bovine mammary epithelial cells challenged with lipoteichoic acid.

Mastitis is a common disease of the dairy cattle, which affects the development of the dairy industry and leads to huge economic losses. Forsythoside A (FTA) has anti-inflammatory, antioxidant, antiviral and anti-apoptotic effects. However, the therapeutic effect and molecular mechanism of FTA on dairy cow mastitis remain unclear. In this study, bovine mammary epithelial cells (BMECs) were stimulated with lipoteichoic acid (LTA), a key virulence factor of Staphylococcus aureus (S. aureus), to construct in vitro models, and then treated with FTA. Subsequently, the differentially expressed genes (DEGs) in different groups were determined by RNA sequencing (RNA-Seq) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyse the possible function of the DEGs, real-time quantitative PCR (RT-qPCR) was used to verify whether the expression levels of these DEGs were consistent with RNA-Seq results. The results showed that cell division cycle 20B (CDC20B), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR), complement factor H-related 5 (CFHR5) and phospholipase A2 group IVA (PLA2G4A) were down-regulated after FTA treatment. In contrast, Kruppel-like factor 15 (KLF15) and Metallothionein 1E (MT1E) were up-regulated. These DEGs are involved in processes such as apoptosis, inflammation and development of cancer. This study provides valuable insights into the transcriptome changes in BMECs after FTA treatment. Further analysis may help identify the underlying molecular mechanisms.

Probing Antibacterial and Anticancer Potential of Selenicereus undatus, Pistacia vera L. and Olea europaea L. against Uropathogens, MCF-7 and A2780 Cancer Cells.

Urinary tract infection is an infectious disease that requires immediate treatment. It can occur in any age group and involves both genders equally. The present study was to check the resistance of some antibiotics and to assess the antibacterial potential of three extracts of three plants against notorious bacteria involved in urinary tract infections. Along with assessing the antibacterial activity of plant extracts, we checked for the anticancer potential of these extracts against the cancer cell lines MCF-7 and A2780. Cancer is the leading cause of mortality in developed countries. Determinations of total flavonoid content, total phenolic content, total alkaloid content, total tannin content, total carotenoid content, and total steroid content were performed. The disk diffusion method was used to analyze the antibacterial activity of plant extracts. Ethanolic extract of Selenicereus undatus showed sensitivity (25-28 mm) against bacteria, whereas chloroform and hexane extracts showed resistance against all bacteria except Staphylococcus (25 mm). Ethanolic extract of Pistacia vera L. showed sensitivity (22-25 mm) against bacteria, whereas chloroform and hexane extracts showed resistance. Ethanolic extract of Olea europaea L. showed sensitivity (8-16 mm) against all bacteria except Staphylococcus, whereas chloroform and hexane extracts showed resistance. Positive controls showed variable zones of inhibition (2-60 mm), and negative control showed 0-1 mm. The antibiotic resistance was much more prominent in the case of hexane and chloroform extracts of all plants, whereas ethanolic extract showed a sensitivity of bacteria against extracts. Both cell lines, MCF-7 and A2780, displayed decreased live cells when treated with plant extracts.

Lactobacillus paracasei TK1501 fermented soybeans alleviate dextran sulfate sodium-induced colitis by regulating intestinal cell function.

BACKGROUND: Probiotic food provide health benefits by regulating intestinal floras via live bacteria, but the shelf life is short and the preservation condition is demanding due to the bacteria being fragile. Owing to these problems, we have tried to find a fermented food that is helpful for inflammatory bowel disease treatment but independent of live bacteria. In addition, the mechanisms of fermented food were investigated. Dextran sulfate sodium was used to model inflammatory bowel disease in mice, and Lactobacillus paracasei TK1501 fermented soybeans and their metabolites were used to treat inflammatory bowel disease. RESULTS: In this study, TK1501 fermented soybean alleviated colitis. However, the efficacy was associated with bacterial metabolites but not live or dead bacteria. Compositional analysis of soybean before and after fermentation shows that soy carbohydrates were used for bacteria growth and produced functional substances. Further, we display the main active ingredient was lipoteichoic acid and peptidoglycan, because lipoteichoic acid reduced the colonic macrophage and peptidoglycan may increase the mucin-2 expression. A cell experiment displayed that lipoteichoic acid could enhance the phagocytosis of macrophages. CONCLUSION: In general, TK1501 fermented soybean alleviating colitis is dependent on metabolites of TK1501, particularly lipoteichoic acid and peptidoglycan. The fermented food may have a long shelf life and lax storage condition. © 2023 Society of Chemical Industry.

Analysis of differentially expressed microRNAs in bovine mammary epithelial cells treated with lipoteichoic acid.

Mastitis is one of the most common diseases of dairy cattle and can be caused by physical stress, chemicals and microbial infection. Staphylococcus aureus is the most common pathogens that induce mastitis in dairy cattle. In this study, bovine mammary epithelial cells (BMECs) were treated either with lipoteichoic acid (LTA, 30 µg/ml) or 1 × phosphate-buffer saline (PBS, control) and RNA-Seq was applied to explore the effect of LTA on the expression microRNAs (miRNAs) in BMECs. Compared to the control group, 43 miRNAs were significantly up-regulated and eight miRNAs were significantly down-regulated. Additionally, 724 genes were significantly up-regulated and 13 genes were significantly down-regulated in LTA group relative to the control group. Bta-miR-196a, bta-miR-2285aj-5p, bta-miR-143, bta-miR-2433, bta-miR-2284f and bta-miR-2368-3p were selected from 51 differentially expressed miRNAs and are discussed in this manuscript. Target gene prediction revealed that the target genes of these six miRNAs were all differentially expressed, including MT1E, SPDYA, FGL1, TLR2, PAPOLG, ZDHHC17 and SMC4. Subsequently, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the target genes with differentially expressed miRNAs were enriched in mitogen-activated protein kinase (MAPK) signalling pathway, rheumatoid arthritis and cancer. Therefore, the results of this study provided new evidences for the molecular mechanism of LTA-induced mastitis, which may provide new targets for the diagnosis and treatment of mastitis in dairy cattle.

Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit.

Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

Spatial regulation of protein A in Staphylococcus aureus.

Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.