Identification of Fur in Pasteurella multocida and the Potential of Its Mutant as an Attenuated Live Vaccine

Pasteurella multocida is a pathogenic microorganism that causes a variety of serious diseases in humans and animals worldwide. The global regulator gene, fur, plays an important role in pathogenesis and regulates the virulence of many bacteria. Here, we identified a fur gene in P. multocida by complementing a Salmonella Choleraesuis Δfur mutant, and characterized a fur mutant strain of P. multocida. The P. multocida Δfur mutant strain exhibited no significant differences in growth and outer membrane protein (OMP) profiles when the complemented strain was compared to the parent. Ducks were used as the model organism to determine the virulence and protection efficacy induced by Δfur mutant strain. Animal experiments showed that colonization by the mutant was decreased by oral infection of live Δfur mutant strain. The LD50 of the ducks infected with the Δfur mutant was 146-fold higher than that of the ducks infected with the wild-type strain when administered through the oral route. Evaluation of the immunogenicity and protective efficacy of the Δfur mutant of P. multocida revealed strong serum IgY and bile IgA immune responses following oral inoculation with the Δfur strain. Ducks that were orally inoculated with the Δfur mutant strain demonstrated 62% protection efficacy against severe lethal challenge with the wild-type P. multocida. This study provides new insights into P. multocida virulence and the potential use of an attenuated vaccine against P. multocida

O-Serotype Conversion in Salmonella Typhimurium Induces Protective Immune Responses against Invasive Non-Typhoidal Salmonella Infections

Salmonella infections remain a big problem worldwide, causing enteric fever by Salmonella Typhi (or Paratyphi) or self-limiting gastroenteritis by non-typhoidal Salmonella (NTS) in healthy individuals. NTS may become invasive and cause septicemia in elderly or immuno-compromised individuals, leading to high mortality and morbidity. No vaccines are currently available for preventing NTS infection in human. As these invasive NTS are restricted to several O-antigen serogroups including B1, D1, C1, and C2, O-antigen polysaccharide is believed to be a good target for vaccine development. In this study, a strategy of O-serotype conversion was investigated to develop live attenuated S. Typhimurium vaccines against the major serovars of NTS infections. The immunodominant O4 serotype of S. Typhimurium was converted into O9, O7, and O8 serotypes through unmarked chromosomal deletion–insertion mutations. O-serotype conversion was confirmed by LPS silver staining and western blotting. All O-serotype conversion mutations were successfully introduced into the live attenuated S. Typhimurium vaccine S738 (Δcrp Δcya) to evaluate their immunogenicity in mice model. The vaccine candidates induced high amounts of heterologous O-polysaccharide-specific functional IgG responses. Vaccinated mice survived a challenge of 100 times the 50% lethality dose (LD50) of wild-type S. Typhimurium. Protective efficacy against heterologous virulent Salmonella challenges was highly O-serotype related. Furthermore, broad-spectrum protection against S. Typhimurium, S. Enteritidis, and S. Choleraesuis was observed by co-vaccination of O9 and O7 O-serotype-converted vaccine candidates. This study highlights the strategy of expressing heterologous O-polysaccharides via genetic engineering in developing live attenuated S. Typhimurium vaccines against NTS infections

Attenuated Salmonella Typhimurium expressing Salmonella Paratyphoid A O-antigen induces protective immune responses against two Salmonella strains

Salmonella enterica serovar Paratyphi A is the main causative agent of paratyphoid fever in many Asian countries. As paratyphoid is spread by the fecal-oral route, the most effective means of controlling S. Paratyphi A infection is through the availability of clean water supplies and working sanitation services. Because sanitation facilities improve slowly in these poor areas and antibiotic resistance is severe, the development of a safe and effective vaccine remains a priority for controlling the spread of paratyphoid disease. In this study, we investigated the strategy of heterologous O-antigenic O2 serotype (S. Paratyphi A characterized) conversion in S. Typhimurium to prevent paratyphoid infections. A series of S. Typhimurium mutants were constructed with replacement of abe, wzxB1 and wbaVB1 genes with respective prt-tyvA1, wzxA1 and wbaVA1, and the results showed that only three genes including prt, wbaVA1 and wzxA1 from S. Paratyphi A presence enable S. Typhimurium to sufficiently express O2 antigen polysaccharide. We also constructed a series of live attenuated S. Typhimurium vaccine candidates expressing heterologous O2 O-antigens, and a mouse model was used to evaluate the immunogenicity of live vaccines. ELISA data showed that vaccine candidates could induce a comparatively high level of S. Paratyphi A and/or S. Typhimurium LPS-specific IgG and IgA responses in murine model, and IgG2a levels were consistently higher than IgG1 levels. Moreover, the functional properties of serum antibodies were evaluated using in vitro C3 complement deposition and opsonophagocytic assays. Our work highlights the potential for developing S. Typhimurium live vaccines against S. Paratyphi A

Regulated Delayed Expression of rfaH in an Attenuated Salmonella enterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen▿

RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent Streptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241

Identification of the Avian Pasteurella multocida phoP Gene and Evaluation of the Effects of phoP Deletion on Virulence and Immunogenicity

Pasteurella multocida (P. multocida) is an animal pathogen of worldwide economic significance that causes fowl cholera in poultry and wild birds. Global gene regulators, including PhoP are important in regulating bacterial virulence and are good targets for developing attenuated vaccines against many pathogenic bacteria. However, the biological significance of phoP gene has not been identified in P. multocida. Here, we identified the phoP gene in P. multocida, and we evaluated the roles of phoP in P. multocida by deleting the phoP gene. The P. multocida phoP mutant exhibited similar growth curves and lipopolysaccharide and outer membrane protein profiles but displayed defective polymyxin resistance in vitro compared with the parent strain. Additionally, the phoP deletion resulted in decreased virulence. The LD50 of the ΔphoP mutant was 32- and 154-fold higher than the parent strain via the oral and intranasal routes, respectively. Transcriptome sequencing analysis showed that 161 genes were up-regulated and 173 genes were down-regulated in the absence of the phoP gene. Finally, the immunogenicity and protective efficacy of the ΔphoP mutant were evaluated. Immunized ducks produced significantly higher levels of serum IgY and bile IgA compared to the control ducks, and immunization with the ΔphoP mutant conferred 54.5% protection efficiency against challenge with the virulent P. multocida. This work provides a platform to dissect the function of phoP and develop a new vaccine against P. multocida

Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure

Escherichia coli BL21 (DE3) and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS). LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004) carrying plasmid pQK004 (pagL and lpxE) produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL) as vaccine adjuvants

Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

Lipopolysaccharide (LPS) is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium). Outer membrane proteins (OMPs) from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46) induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa

Construction and Evaluation of an Efficient Live Attenuated <i>Salmonella</i> Choleraesuis Vaccine and Its Ability as a Vaccine Carrier to Deliver Heterologous Antigens

The gram-negative facultative intracellular pathogen Salmonella enterica serotype Choleraesuis, also known as S. Choleraesuis, is a major financial loss for the pig business. C500 is a vaccine strain that has been used for preventing S. Choleraesuis infection in pigs for many years in China. Although it possessed good immunogenicity and protection efficacy, it still showed severe side effects. The truncation of the key gene rpoS in C500 was believed to take the major responsibility for its attenuation. To achieve a good balance between attenuation and immunogenicity, rpoS was restored to an active state, and other essential virulent genes of crp, fur, phoP, and aroA were evaluated for their effects of deletion on safety and immunogenicity. Animal experiments demonstrated that C5001 (C500 rpoS+ Δcrp10) and C5002 (C500 rpoS+ Δfur9) showed an excellent ability to induce an immune response. To further decrease the endotoxic activity, the combination mutations of ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9 were introduced into the mutant strains to generate 1′-dephosphorylated lipid A. Animal experiments showed that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) induced higher levels of IgG and secreted IgA antibodies and provided a higher protection rate than SC1 (C500 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) and SC2 (C500 rpoS+ Δcrp10 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9). We also evaluated the ability of SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) as a vaccine carrier to deliver heterologous protein antigens and polysaccharide antigens. The results indicated that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) showed an excellent ability to deliver heterologous antigens and induce the host to produce high levels of antibodies. Together, these results indicate that we constructed a safe and efficient attenuated strain of the S. Choleraesuis vaccine, which demonstrated strong resistance to infection with wild-type S. Choleraesuis and can be employed as a universal vector for the delivery of recombinant antigens