Trueperella pyogenes is a commensal and opportunistic pathogen that normally causes mastitis, liver abscesses and pneumonia of economically important livestock. To develop efficacious and potent vaccine against T. pyogenes, chimeric gene DNA vaccines were constructed and encapsulated in chitosan nanoparticles (pPCFN-CpG-CS-NPs). The pPCFN-CpG-CS-NPs consists of the plo, cbpA, fimA, and nanH gene of T. pyogenes and CpG ODN1826. It was produced with good morphology, high stability, a mean diameter of 93.58 nm, and a zeta potential of + 5.27 mV. Additionally, chitosan encapsulation was confirmed to protect the DNA plasmid from DNase I digestion. The immunofluorescence assay indicated that the four-chimeric gene could synchronously express in HEK293T cells and maintain good bioactivity. Compared to the mice immunized with the control plasmid, in vivo immunization showed that mice immunized with the pPCFN-CpG-CS-NPs had better immune responses, and release of the plasmid DNA was prolonged. Importantly, immunization with pPCFN-CpG-CS-NPs could significantly protect mice from highly virulent T. pyogenes TP7 infection. This study indicates that chitosan-DNA nanoparticles are potent immunization candidates against T. pyogenes infection and provides strategies for the further development of novel vaccines encapsulated in chitosan nanoparticles.

Microbial communities are susceptible to the public goods dilemma, whereby individuals can gain an advantage within a group by utilizing, but not sharing the cost of producing, public goods. In bacteria, the development of quorum sensing (QS) can establish a cooperation system in a population by coordinating the production of costly and sharable extracellular products (public goods). Cooperators with intact QS system and robust ability in producing public goods are vulnerable to being undermined by QS-deficient defectors that escape from QS but benefit from the cooperation of others. Although microorganisms have evolved several mechanisms to resist cheating invasion in the public goods game, it is not clear why cooperators frequently coexist with defectors and how they form a relatively stable equilibrium during evolution. We show that in Pseudomonas aeruginosa, QS-directed social cooperation can select a conditional defection strategy prior to the emergence of QS-mutant defectors, depending on resource availability. Conditional defectors represent a QS-inactive state of wild type (cooperator) individual and can invade QS-activated cooperators by adopting a cheating strategy, and then revert to cooperating when there are abundant nutrient supplies irrespective of the exploitation of QS-mutant defector. Our mathematical modeling further demonstrates that the incorporation of conditional defection strategy into the framework of iterated public goods game with sound punishment mechanism can lead to the coexistence of cooperator, conditional defector, and defector in a rock-paper-scissors dynamics. These findings highlight the importance of behavioral heterogeneity in stabilizing the population structure and provide a potential reasonable explanation for the maintenance and evolution of cooperation in microbial communities.

Klebsiella pneumoniae is an important gram-negative bacterium that causes severe respiratory and healthcare-associated infections. Although antibiotic therapy is applied to treat severe infections caused by K. pneumoniae, drug-resistant isolates pose a huge challenge to clinical practices owing to adverse reactions and the mismanagement of antibiotics. Several studies have attempted to develop vaccines against K. pneumoniae, but there are no licensed vaccines available for the control of K. pneumoniae infection. In the current study, we constructed a novel DNA vaccine, pVAX1-YidR, which encodes a highly conserved virulence factor YidR and a recombinant expression plasmid pVAX1-IL-17 encoding Interleukin-17 (IL-17) as a molecular adjuvant. Adaptive immune responses were assessed in immunized mice to compare the immunogenicity of the different vaccine schemes. The results showed that the targeted antigen gene was expressed in HEK293T cells using an immunofluorescence assay. Mice immunized with pVAX1-YidR elicited a high level of antibodies, induced strong cellular immune responses, and protected mice from K. pneumoniae challenge. Notably, co-immunization with pVAX1-YidR and pVAX1-IL-17 significantly augmented host adaptive immune responses and provided better protection against K. pneumoniae infections in vaccinated mice. Our study demonstrates that combined DNA vaccines and molecular adjuvants is a promising strategy to develop efficacious antibacterial vaccines against K. pneumoniae infections.

Klebsiella pneumoniae is a prominent Gram-negative and encapsulated opportunistic pathogen that causes a multitude of infections such as severe respiratory and healthcare-associated infections. Despite the widespread anti-microbial resistance and the high mortality rate, currently, no clinically vaccine is approved for battling K. pneumoniae . To date, messenger RNA (mRNA) vaccine is one of the most advancing technologies and are extensively investigated for viral infection, while infrequently applied for prevention of bacterial infections. In the present study, we aim to construct a new mRNA vaccine encoding YidR or combining with a tissue plasminogen activator signal sequence for preventing K. pneumoniae infection. Adaptive immunity was determined in mRNA vaccines-immunized mice and the protective effects of mRNA vaccines were evaluated in K. pneumoniae infected models. The results showed that lipid nanoparticle (LNP)-YidR-mRNA vaccine was produced with good morphology, high the encapsulation efficiency, and the specific antigen was highly expressed in cells in vitro . In addition, immunization with either LNP-YidR or LNP-YidR-SP elicited a Th1-biased immune response, reduced bacterial load, and provided broad protection in the lung infection models. Importantly, the LNP-YidR-SP mRNA vaccine induced strong adaptive humoral and cellular immunity and increased the survivability of mice compared to the other groups. Our findings serve as a focal point for developing a potential mRNA vaccine against K. pneumoniae , indicating the potential of mRNA vaccines for improving next-generation bacterial vaccine. IMPORTANCE: K. pneumoniae is a notorious and clinical bacterium that is evolving in community-acquired and nosocomial settings. This opportunistic pathogen causes severe infectious diseases, including urinary tract infection and pneumonia, and causes a concerning global public burden. Despite efforts having been created to develop different types of K. pneumoniae vaccines, there is no licensed vaccine for preventing K. pneumoniae infection. Therefore, to develop an effective tactic is essential to combat K. pneumoniae -caused diseases. This study provides a novel vaccine strategy against K. pneumoniae and a potent platform to elicit high levels of humoral and cell-meditated immunity.

The emergence of antibiotic resistant Gram-negative bacteria such as Klebsiella pneumoniae (KP) is becoming a major public health threat and imposing a financial burden worldwide. A serious lack of new drugs under development is undermining efforts to fight them. In this study, we report a potent combination of linezolid and polymyxin B nonapeptide PBNP (LP) against KP infection in vitro and in vivo. The checkerboard test and the time-kill assay were performed to detect the antibacterial activity of LP against KP in vitro. And the Caenorhabditis elegans (C. elegans) was used as infection model to evaluate the protective effect of LP against KP infection in vivo. The LP combination showed significantly synergistic activity and antibacterial effects against KP, while linezolid and PBNP as monotherapies revealed no dramatically antibacterial activity against the KP strains. Additionally, we found that the LP treatment altered the biofilm production and morphology of KP. Furthermore, the LP treatments significantly protected C. elegans from KP infection. In conclusion, this study indicated that the LP combination exhibited significantly synergistic activity against KP and PBNP can be used as a potential activity enhancer. More importantly, this strategy provided the improvement of antibacterial activity spectrum of agents like linezolid and represented a potent alternative to overcome antibiotic resistance in the future.