IgG1 Is Required for Optimal Protection after Immunization with the Purified Porin OmpD from Salmonella Typhimurium.

In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Salmonella Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as S. Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified S. Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to S. Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as S. Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.

Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium.

The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S Typhimurium to immune serum identified a repertoire of S Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

Identification of a common immune signature in murine and human systemic Salmonellosis.

Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined. We have previously shown evidence for antibody-mediating protection against invasive Salmonellosis in mice and African children. To generate an overview of antibody targeting in systemic Salmonellosis, a Salmonella proteomic array containing over 2,700 proteins was constructed and probed with immune sera from Salmonella-infected mice and humans. Analysis of multiple inbred mouse strains identified 117 antigens recognized by systemic antibody responses in murine Salmonellosis. Importantly, many of these antigens were independently identified as target antigens using sera from Malawian children with Salmonella bacteremia, validating the study of the murine model. Furthermore, vaccination with SseB, the most prominent antigenic target in Malawian children, provided mice with significant protection against Salmonella infection. Together, these data uncover an overlapping immune signature of disseminated Salmonellosis in mice and humans and provide a foundation for the generation of a protective subunit vaccine.

Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Salmonella Typhimurium ST313.

Background: Salmonella Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal Salmonellae is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in S. Typhimurium ST313. Methods: Six S. Typhimurium ST313 bloodstream isolates, three of which were antibody resistant, were studied. Genomic content (single nucleotide polymorphisms and larger chromosomal modifications) of the strains was determined by Illumina and PACBIO sequencing, and functionally characterized using RNA-seq, transposon directed insertion site sequencing (TraDIS), targeted gene deletion and transfer of selected point mutations in an attempt to identify features associated with serum resistance.   Results: Sequence polymorphisms in genes from strains with atypical serum susceptibility when transferred from strains that were highly resistant or susceptible to a strain that exhibited intermediate susceptibility did not significantly alter serum killing phenotype. No large chromosomal modifications typified serum resistance or susceptibility. Genes required for resistance to serum identified by TraDIS and RNA-seq included those involved in exopolysaccharide synthesis, iron scavenging and metabolism. Most of the down-regulated genes were associated with membrane proteins. Resistant and susceptible strains had distinct transcriptional responses to serum, particularly related to genes responsible for polysaccharide biosynthesis. There was higher upregulation of wca locus genes, involved in the biosynthesis of colanic acid exopolysaccharide, in susceptible strains and increased expression of fepE, a regulator of very long-chain lipopolysaccharide in resistant strains. Conclusion: Clinical isolates of S. Typhimurium ST313 exhibit distinct antibody susceptibility phenotypes that may be associated with changes in gene expression on exposure to serum.

Cytokine Profiles during Invasive Nontyphoidal Salmonella Disease Predict Outcome in African Children.

Nontyphoidal Salmonella is a leading cause of sepsis in African children. Cytokine responses are central to the pathophysiology of sepsis and predict sepsis outcome in other settings. In this study, we investigated cytokine responses to invasive nontyphoidal Salmonella (iNTS) disease in Malawian children. We determined serum concentrations of 48 cytokines with multiplexed immunoassays in Malawian children during acute iNTS disease (n = 111) and in convalescence (n = 77). Principal component analysis and logistic regression were used to identify cytokine signatures of acute iNTS disease. We further investigated whether these responses are altered by HIV coinfection or severe malnutrition and whether cytokine responses predict inpatient mortality. Cytokine changes in acute iNTS disease were associated with two distinct cytokine signatures. The first is characterized by increased concentrations of mediators known to be associated with macrophage function, and the second is characterized by raised pro- and anti-inflammatory cytokines typical of responses reported in sepsis secondary to diverse pathogens. These cytokine responses were largely unaltered by either severe malnutrition or HIV coinfection. Children with fatal disease had a distinctive cytokine profile, characterized by raised mediators known to be associated with neutrophil function. In conclusion, cytokine responses to acute iNTS infection in Malawian children are reflective of both the cytokine storm typical of sepsis secondary to diverse pathogens and the intramacrophage replicative niche of NTS. The cytokine profile predictive of fatal disease supports a key role of neutrophils in the pathogenesis of NTS sepsis.

Emergence of host-adapted Salmonella Enteritidis through rapid evolution in an immunocompromised host.

Host adaptation is a key factor contributing to the emergence of new bacterial, viral and parasitic pathogens. Many pathogens are considered promiscuous because they cause disease across a range of host species, while others are host-adapted, infecting particular hosts(1). Host adaptation can potentially progress to host restriction, where the pathogen is strictly limited to a single host species and is frequently associated with more severe symptoms. Host-adapted and host-restricted bacterial clades evolve from within a broader host-promiscuous species and sometimes target different niches within their specialist hosts, such as adapting from a mucosal to a systemic lifestyle. Genome degradation, marked by gene inactivation and deletion, is a key feature of host adaptation, although the triggers initiating genome degradation are not well understood. Here, we show that a chronic systemic non-typhoidal Salmonella infection in an immunocompromised human patient resulted in genome degradation targeting genes that are expendable for a systemic lifestyle. We present a genome-based investigation of a recurrent blood-borne Salmonella enterica serotype Enteritidis (S. Enteritidis) infection covering 15 years in an interleukin-12 ß1 receptor-deficient individual that developed into an asymptomatic chronic infection. The infecting S. Enteritidis harboured a mutation in the mismatch repair gene mutS that accelerated the genomic mutation rate. Phylogenetic analysis and phenotyping of multiple patient isolates provides evidence for a remarkable level of within-host evolution that parallels genome changes present in successful host-restricted bacterial pathogens but never before observed on this timescale. Our analysis identifies common pathways of host adaptation and demonstrates the role that immunocompromised individuals can play in this process.

Emergence of host-adapted Salmonella Enteritidis through rapid evolution in an immunocompromised host.

Host adaptation is a key factor contributing to the emergence of new bacterial, viral and parasitic pathogens. Many pathogens are considered promiscuous because they cause disease across a range of host species, while others are host-adapted, infecting particular hosts1. Host adaptation can potentially progress to host restriction where the pathogen is strictly limited to a single host species and is frequently associated with more severe symptoms. Host-adapted and host-restricted bacterial clades evolve from within a broader host-promiscuous species and sometimes target different niches within their specialist hosts, such as adapting from a mucosal to a systemic lifestyle. Genome degradation, marked by gene inactivation and deletion, is a key feature of host adaptation, although the triggers initiating genome degradation are not well understood. Here, we show that a chronic systemic non-typhoidal Salmonella infection in an immunocompromised human patient resulted in genome degradation targeting genes that are expendable for a systemic lifestyle. We present a genome-based investigation of a recurrent blood-borne Salmonella enterica serotype Enteritidis (S. Enteritidis) infection covering 15 years in an interleukin (IL)-12 ß-1 receptor-deficient individual that developed into an asymptomatic chronic infection. The infecting S. Enteritidis harbored a mutation in the mismatch repair gene mutS that accelerated the genomic mutation rate. Phylogenetic analysis and phenotyping of multiple patient isolates provides evidence for a remarkable level of within-host evolution that parallels genome changes present in successful host-restricted bacterial pathogens but never before observed on this timescale. Our analysis identifies common pathways of host adaptation and demonstrates the role that immunocompromised individuals can play in this process.

Increased severity of respiratory infections associated with elevated anti-LPS IgG2 which inhibits serum bactericidal killing.

Although specific antibody induced by pathogens or vaccines is a key component of protection against infectious threats, some viruses, such as dengue, induce antibody that enhances the development of infection. In contrast, antibody-dependent enhancement of bacterial infection is largely unrecognized. Here, we demonstrate that in a significant portion of patients with bronchiectasis and Pseudomonas aeruginosa lung infection, antibody can protect the bacterium from complement-mediated killing. Strains that resist antibody-induced, complement-mediated killing produce lipopolysaccharide containing O-antigen. The inhibition of antibody-mediated killing is caused by excess production of O-antigen-specific IgG2 antibodies. Depletion of IgG2 to O-antigen restores the ability of sera to kill strains with long-chain O-antigen. Patients with impaired serum-mediated killing of P. aeruginosa by IgG2 have poorer respiratory function than infected patients who do not produce inhibitory antibody. We suggest that excessive binding of IgG2 to O-antigen shields the bacterium from other antibodies that can induce complement-mediated killing of bacteria. As there is significant sharing of O-antigen structure between different Gram-negative bacteria, this IgG2-mediated impairment of killing may operate in other Gram-negative infections. These findings have marked implications for our understanding of protection generated by natural infection and for the design of vaccines, which should avoid inducing such blocking antibodies.