KbvR mutant of Klebsiella pneumoniae affects the synthesis of type 1 fimbriae and provides protection to mice as a live attenuated vaccine.

Klebsiella pneumoniae is a leading cause of severe infections in humans and animals, and the emergence of multidrug-resistant strains highlights the need to develop effective vaccines for preventing such infections. Live attenuated vaccines are attractive vaccine candidates available in the veterinary field. We recently characterized that the K. pneumoniae kbvR (Klebsiella biofilm and virulence regulator) mutant was a highly attenuated strain in the mice model. In the present study, the characterization, safety, and protective efficacy of ΔkbvR strain as a live attenuated vaccine were evaluated. The synthesis and activity of type 1 fimbriae were increased in the ΔkbvR strain. All mice inoculated by the subcutaneous route with 105, 106, and 107 colony-forming units (CFU) doses of the ΔkbvR strain survived. Subcutaneous immunization with two doses of 105 or 107 CFU ΔkbvR elicited a robust humoral immune response, and provided protection against the following K. pneumoniae intraperitoneal infection. The antisera of mice immunized with 105 CFU dose improved the opsonophagocytic ability and complement-mediated lysis not only to the same serotype strain but also to the different serotype strain. The passive transfer of antisera from 105 CFU dose-immunized mice provided protection against K. pneumoniae infection. Overall, our results suggest the great potential of the ΔkbvR strain as a novel vaccine candidate against K. pneumoniae infections in herds or humans.

High Osmotic Stress Increases OmpK36 Expression through the Regulation of KbvR to Decrease the Antimicrobial Resistance of Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen known for its high frequency of antimicrobial resistance. Responses to various environmental stresses during its life can influence the resistance to antibiotics. Here, we demonstrate the role and mechanism of KbvR regulator in the response to environmental osmotic stress and in the effect of osmotic stress on antimicrobial resistance. The kbvR mutant strain exhibited increasing tolerance to high osmotic stress and certain antibiotics, including ß-lactams. The expression levels of KbvR and outer membrane porin OmpK36 were upregulated in response to high osmotic stress in the wild type (WT), and the deletion of kbvR decreased the expression level of ompK36. The membrane permeability of the kbvR mutant strain was decreased, which was partly restored through the upregulated expression of OmpK36. The DNA affinity purification sequencing (DAP-seq) and microscale thermophoresis (MST) assay disclosed the binding of KbvR to the promoter of the ompK36 gene, indicating that KbvR directly and positively regulated the expression of OmpK36. The high osmotic stress increased the susceptibility to ß-lactams and the expression of ompK36 in the WT strain. However, the increased ompK36 expression and the susceptibility to ß-lactams in the kbvR mutant strain under high osmotic stress were lower than those of WT. In conclusion, our study has identified that high osmotic stress in the environment influenced the resistance of K. pneumoniae to antibiotics and that the regulation of KbvR with OmpR on the expression of OmpK36 was involved in countering high osmotic stress to change the antimicrobial resistance. IMPORTANCE Klebsiella pneumoniae is considered a global threat because of the rising prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. The sensing and adaption abilities of bacteria to the environmental osmotic stress can change the expression of their outer membrane porins, membrane permeability, and resistance to antibiotics. This study reports that KbvR is a newly found regulator that can be upregulated under high osmotic stress and directly regulate the expression of OmpK36 to change the resistance of K. pneumoniae to ß-lactam antibiotics. The results demonstrate how adaptation to high osmotic stress changes the sensitivity of K. pneumoniae to antibiotics. The mechanism can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints in governing adaptation to diverse environmental challenges.

The KbvR Regulator Contributes to Capsule Production, Outer Membrane Protein Biosynthesis, Antiphagocytosis, and Virulence in Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that mostly affects patients with weakened immune systems, but a few serotypes (especially K1 and K2) are highly invasive and result in systemic infection in healthy persons. The ability to evade and survive the components of the innate immune system is critical in infection. To investigate the role and mechanism of transcription regulator KP1_RS12260 (KbvR) in virulence and defense against the innate immune response, kbvR deletion mutant and complement strains were constructed. The in vivo animal infection assay and in vitro antiphagocytosis assay demonstrate K. pneumoniae KbvR is an important regulator that contributes to virulence and the defense against phagocytosis of macrophages. The transcriptome analysis and phenotype experiments demonstrated that deletion of kbvR decreased production of capsular polysaccharide (CPS) and biosynthesis of partly outer membrane proteins (OMPs). The findings suggest that KbvR is a global regulator that confers pathoadaptive phenotypes, which provide several implications for improving our understanding of the pathogenesis of K. pneumoniae.

Identification of two missense mutations of ERCC6 in three Chinese sisters with Cockayne syndrome by whole exome sequencing.

Cockayne syndrome (CS) is a rare autosomal recessive disorder, the primary manifestations of which are poor growth and neurologic abnormality. Mutations of the ERCC6 and ERCC8 genes are the predominant cause of Cockayne syndrome, and the ERCC6 gene mutation is present in approximately 65% of cases. The present report describes a case of Cockayne syndrome in a Chinese family, with the patients carrying two missense mutations (c.1595A>G, p.Asp532Gly and c.1607T>G, p.Leu536Trp) in the ERCC6 gene in an apparently compound heterozygote status, especially, p.Asp532Gly has never been reported. The compound heterozygote mutation was found in three patients in the family using whole exome sequencing. The patients' father and mother carried a heterozygous allele at different locations of the ERCC6 gene, which was confirmed by Sanger DNA sequencing. The two mutations are both located in the highly conserved motif I of ATP-binding helicase and are considered "Damaging," "Probably Damaging," "Disease Causing," and "Conserved", indicating the role of DNA damage in the pathogenetic process of the disease. The results not only enrich the ERCC6 mutations database, but also indicate that whole exome sequencing will be a powerful tool for discovering the disease causing mutations in clinical diagnosis.