Klebsiella pneumoniae (K. pneumoniae) is a gram-negative conditionally pathogenic bacterium that causes disease primarily in immunocompromised individuals. Recently, highly virulent K. pneumoniae strains have caused severe disease in healthy individuals, posing significant challenges to global infection control. Capsular polysaccharide (CPS), a major virulence determinant of K. pneumoniae, protects the bacteria from being killed by the host immune system, suggesting an urgent need for the development of drugs to prevent or treat K. pneumoniae infections. In this study, BY3 compounded traditional Chinese medicine residue (TCMR) was carried out using Lactobacillus rhamnosus as a fermentation strain, and BY3 compounded TCMR fermentation broth (BY3 fermentation broth) was obtained. The transcription of K. pneumoniae CPS-related biosynthesis genes after treatment with BY3 fermentation broth was detected using quantitative real-time polymerase chain reaction. The effects of BY3 fermentation broth on K. pneumoniae serum killing, macrophage phagocytosis, complement deposition and human β-defensin transcription were investigated. The therapeutic effect of BY3 fermentation broth on K. pneumoniae-infected mice was also observed, and the major active components of BY3 fermentation broth were analysed via LC‒MS analysis, network pharmacology, and molecular docking. The results showed that BY3 fermentation broth inhibited K. pneumoniae CPS production and downregulated transcription of CPS-related biosynthesis genes, which weakened bacterial resistance to serum killing and phagocytosis, while promoting bacterial surface complement C3 deposition and human β-defensin expression. BY3 fermentation broth demonstrated safety and therapeutic effects in vivo and in vitro, restoring body weight and visceral indices, significantly reducing the organ bacterial load and serum cytokine levels, and alleviating pathological organ damage in mice. In addition, three natural compounds-oleanolic acid, quercetin, and palmitoleic acid-were identified as the major active components in the BY3 fermentation broth. Therefore, BY3 fermentation broth may be a promising strategy for the prevention or treatment of K. pneumoniae infections.

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages.
OBJECTIVE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.

Capsular polysaccharide (CPS) as a probiotic component has the ability to regulate the function of the host\'s immune system. However, how the structure and function of heat-killed CPS are altered remains unclear. In the present study, CPS were isolated and purified from live (LCPS) and heat-killed (HCPS) Lacticaseibacillus paracasei 6235. The differences in structure and immunomodulation between LCPS and HCPS were compared and analyzed. The results demonstrate that after heat killed, the molecular weight of CPS decreased from 23.4 kDa to 17.5 kDa, with the disappearance of galactosamine in the monosaccharide composition, and changes in the microstructure. Methylation analysis and nuclear magnetic resonance analysis revealed that the LCPS and HCPS are similar in structure, which main units of →3,4)-α-D-Glcp-(1→4)-α-D-Galp-(1→3)-β-L-Rhap-(1→6)-β-D-Galp-(1→, and repeating units of →3,4)-α-D-Glcp-(1→, →3)-β-L-Rhap-(1→, and →4)-α-D-Galp-(1→ residues. Furthermore, both LCPS and HCPS significantly downregulated the expression of pro-inflammatory cytokines in RAW264.7 cells induced by LPS. Specifically, HCPS reduced the levels of IL-6 and IL-1β by 79.38 % and 88.42 %, respectively, compared to LCPS. Concurrently, both LCPS and HCPS effectively mitigated inflammatory responses through the NF-κB and MAPK signaling pathways. Moreover, compared to LCPS, HCPS increased the protein expression levels of NF-κB/p-NF-κB and IκB/p-IκB by 26.14 % and 28.92 %, respectively. These results suggest that CPS has a role in modulating immune responses and that HCPS is more effective. This study can be further developed into new products related to postbiotics.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Bacteria utilize intercellular communication to orchestrate essential cellular processes, adapt to environmental changes, develop antibiotic tolerance, and enhance virulence. This communication, known as quorum sensing (QS), is mediated by the exchange of small signalling molecules called autoinducers. AI-2 QS, regulated by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase), acts as a universal intercellular communication mechanism across gram-positive and gram-negative bacteria and is crucial for diverse bacterial processes. In this study, we demonstrated that in Streptococcus suis (S. suis), a notable zoonotic pathogen, AI-2 QS enhances galactose utilization, upregulates the Leloir pathway for capsular polysaccharide (CPS) precursor production, and boosts CPS synthesis, leading to increased resistance to macrophage phagocytosis. Additionally, our molecular docking and dynamics simulations suggest that, similar to S. pneumoniae, FruA, a fructose-specific phosphoenolpyruvate phosphotransferase system prevalent in gram-positive pathogens, may also function as an AI-2 membrane surface receptor in S. suis. In conclusion, our study demonstrated the significance of AI-2 in the synthesis of galactose metabolism-dependent CPS in S. suis. Additionally, we conducted a preliminary analysis of the potential role of FruA as a membrane surface receptor for S. suis AI-2.

The role of gut microbiota in host defense against nontuberculous mycobacterial lung disease (NTM-LD) was poorly understood. Here, we showed significant gut microbiota dysbiosis in patients with NTM-LD. Reduced abundance of Prevotella copri was significantly associated with NTM-LD and its disease severity. Compromised TLR2 activation activity in feces and plasma in the NTM-LD patients was highlighted. In the antibiotics-treated mice as a study model, gut microbiota dysbiosis with reduction of TLR2 activation activity in feces, sera, and lung tissue occurred. Transcriptomic analysis demonstrated immunocompromised in lung which were closely associated with increased NTM-LD susceptibility. Oral administration of P. copri or its capsular polysaccharides enhanced TLR2 signaling, restored immune response, and ameliorated NTM-LD susceptibility. Our data highlighted the association of gut microbiota dysbiosis, systematically compromised immunity and NTM-LD development. TLR2 activation by P. copri or its capsular polysaccharides might help prevent NTM-LD.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host\'s ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

The capsular polysaccharides (CPS) of Group B Streptococcus play a crucial role as virulence determinants and are potential candidates for antigenic components in vaccine formulations. Alkaline treatments are commonly used to extract polysaccharides owing to their efficiency and cost-effectiveness; however, they may induce the removal of N-acetyl groups from CPS. This study involved re-N-acetylation of CPS Ia to improve its biological functionality. The structural modifications and enhanced antigenicity of CPS Ia were observed after re-N-acetylation. The tetanus toxoid (TT) was conjugated with either partially de-N-acetylated or fully re-N-acetylated CPS. As a result, the conjugate containing re-N-acetylated CPS (IaReN-TT) enhanced the induction of IgG antibody levels and functional antibodies in mice. Both passive and active protection assays substantiated the superior protective efficacy of IaReN-TT, suggesting that the re-N-acetylation of CPS Ia could be a critical step in refining the immunogenic profile of glycoconjugate vaccines.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP) in goats. Inactivated vaccines and capsular polysaccharide (CPS) indirect hemagglutination reagents are available for prevention and serological detection, but high culture costs and complex antigen quantification have been plagued by production staff. In order to solve these problems in production practice, a sugar fermentation medium with an initial pH value of 7.8, which could improve the production of two antigens simultaneously, was screened out by changing the initial pH value based on previous Mccp metabolomics analysis. Since phenol red can be identified by UV absorption spectrum and cetyltrimethylammonium bromide (CTAB) can bind to anionic capsular polysaccharide, a UV spectrum measurement method for analyzing the culture stage reached by Mccp and a CTAB precipitation test for relative quantification of capsular polysaccharide antigen content in the fermentation broth were established. The UV spectrum observation method can guide the production of Mccp according to the growth curve of Mccp, which greatly reduces the monitoring time of the traditional CCU method and improves the accuracy of the original eye-observation method. The established CTAB precipitation test can complete the monitoring of CPS content within 5 hours, which greatly reduces the time required compared with the traditional differential technique, and its accuracy was verified by the phenol-sulfuric acid method. The optimized culture medium and the two correlation comparison methods established in this study can effectively reduce the production cost of Mccp and improve the production efficiency. The two assays have been used in the research at our laboratory, which provides experimental data for further improvement of the production process of CCPP inactivated vaccine and capsular polysaccharide as well as rapid quantification.