Eleanor S. Metcalf

Absence of All Components of the Flagellar Export and Synthesis Machinery Differentially Alters Virulence of Salmonella enterica Serovar Typhimurium in Models of Typhoid Fever, Survival in Macrophages, Tissue Culture Invasiveness, and Calf Enterocolitis

ABSTRACT In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella entericaserovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.

Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens.

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

ABSTRACT Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC+phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

Defect in macrophage effector function confers Salmonella typhimurium susceptibility on C3H/HeJ mice

Abstract The defective allele of the endotoxin response locus ( Lps d ) renders mice (e.g., C3H/HeJ strain) both endotoxin hyporesponsive and susceptible to Salmonella typhimurium . In this study, the mechanism of Lps d -regulated susceptibility to murine typhoid was examined. C3H/ HeJ mice became significantly more resistant to S. typhimurium by reconstitution with bone marrow from syngeneic C3H/HeN mice ( Lps n , salmonella resistant). Thus, the Lps d resistance defect appeared to reside in a radiosensitive bone marrow-derived cell(s). At least one of the abnormal cell types appeared to be a macrophage because C3H/HeJ mice preinfected with Mycobacterium bovis (BCG) were, in contrast to controls, able to restrict early salmonella replication in their spleens and displayed a signficant increase in mean time to death. In contrast, no deficiency in uptake of salmonellae by C3H/HeJ macrophages was observed. These results indicate that the early deaths of C3H/HeJ mice following S. typhimurium challenge reflect a failure of their macrophages to limit the growth of these gram-negative bacteria.

Infection-Induced Expansion of a MHC Class Ib-Dependent Intestinal Intraepithelial γδ T Cell Subset

Salmonella species invade the host via the intestinal epithelium. Hence, intestinal intraepithelial lymphocytes (iIELs) are potentially the first element of the immune system to encounter Salmonella during infection. In this study, we demonstrate, in a mouse model, the expansion of a CD8αβ+CD94−TCRγδ+ T cell subset within the iIEL population in response to oral infection with virulent or avirulent Salmonella. This population can be detected 3 days following infection, represents up to 15% of the TCRγδ+ iIELs, and is dependent on the MHC class Ib molecule T23 (Qa-1). Qa-1 is expressed by intestinal epithelial cells and thus accessible for iIEL recognition. Such cells may play a role in the early immune response to Salmonella.

The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity

Class I molecules with limited polymorphism have been implicated in the host response to infectious agents. Following infection with Salmonella typhimurium, mice develop a CD8+ CTL response that specifically recognizes bacteria infected cells. An immunodominant component of the CTL response recognizes a peptide epitope derived from the Salmonella GroEL molecule that is presented by the non-polymorphic MHC class Ib molecule Qa-1. T cells recognizing the bacterial peptide also cross-recognize a homologous peptide from the mammalian hsp60 molecule. Since Qa-1 has a functional equivalent in humans, this observation may be relevant not only to the host response involved in clearing infection but also in understanding the link between infection with Gram-negative pathogens and autoimmune disease.

Enterobacterial Common Antigen Mutants of Salmonella enterica Serovar Typhimurium Establish a Persistent Infection and Provide Protection against Subsequent Lethal Challenge

ABSTRACT Infection with Salmonella spp. is a significant source of disease globally. A substantial proportion of these infections are caused by Salmonella enterica serovar Typhimurium. Here, we characterize the role of the enterobacterial common antigen (ECA), a surface glycolipid ubiquitous among enteric bacteria, in S. Typhimurium pathogenesis. Construction of a defined mutation in the UDP-N-acetylglucosamine-1-phosphate transferase gene, wecA, in two clinically relevant strains of S. Typhimurium, TML and SL1344, resulted in strains that were unable to produce ECA. Loss of ECA did not affect the gross cell surface ultrastructure, production of lipopolysaccharide (LPS), flagella, or motility. However, the wecA mutant strains were attenuated in both oral and intraperitoneal mouse models of infection (P < 0.001 for both routes of infection; log rank test), and virulence could be restored by complementation of the wecA gene in trans. Despite the avirulence of the ECA-deficient strains, the wecA mutant strains were able to persistently colonize systemic sites (spleen and liver) at moderate levels for up to 70 days postinfection. Moreover, immunization with the wecA mutant strains provided protection against a subsequent lethal oral or intraperitoneal challenge with wild-type S. Typhimurium. Thus, wecA mutant (ECA-negative) strains of Salmonella may be useful as live attenuated vaccine strains or as vehicles for heterologous antigen expression.

Bacterial and host factors involved in the major histocompatibility complex class Ib-restricted presentation of Salmonella Hsp 60: novel pathway.

ABSTRACT Previously, a peptide epitope derived from the Hsp 60 molecule of Salmonella that is presented by the major histocompatibility complex (MHC) class Ib molecule Qa-1 to CD8+ cytotoxic T cells (CTLs) was described. In the present study we investigated the Salmonella-induced processing and presentation pathway for generating this Qa-1-restricted epitope. Live bacteria and, to a lesser extent, opsonized heat-killed bacteria are able to sensitize target cells for lysis by Salmonella-specific CTL. In contrast, heat-killed bacteria cannot sensitize target cells. Presentation of the Hsp 60 epitope appears independent of bacterial internalization, because cytochalasin D does not affect presentation. Moreover, Salmonella strains defective in the InvA or InvE operon, two critical components of the type III secretion pathway, are as efficient as wild-type Salmonella enterica serovar Typhimurium in sensitizing infected targets to lysis. Collectively, these results suggest the existence of a novel antigen-processing pathway in which exogenous antigens gain access to the cytosolic MHC class I processing machinery. Considering the abundant nature of bacterial Hsp 60 and the upregulation of this protein after Salmonella infection of eukaryotic cells, this mode of antigen presentation may be particularly relevant to understanding the host defense mechanisms against gram-negative bacteria.

An enterobacterial common antigen mutant of Salmonella enterica serovar Typhimurium as a vaccine candidate

Due to increasing rates of invasive Salmonella enterica serovar Typhimurium infection, there is a need for an effective vaccine to prevent this disease. Previous studies showed that a mutation in the first gene of the Enterobacterial common antigen biosynthetic pathway, wecA, resulted in attenuation of S. Typhimurium in a murine model of salmonellosis. Furthermore, immunization with a wecA(-) strain protected against lethal challenge with the parental wild type S. Typhimurium strain. Herein, we examined whether the S. Typhimurium wecA(-) strain could also provide cross-protection against non-parental strains of S. Typhimurium and S. Enteritidis. We found that intraperitoneal immunization (IP) with S. Typhimurium SL1344 wecA(-) resulted in a significant increase in survival compared to control mice for all Salmonella challenge strains tested. Oral immunization with SL1344 wecA(-) also resulted in increased survival; however, protection was less significant than with intraperitoneal immunization. The increase in survival of SL1344 wecA(-) immunized mice was associated with a Salmonella-specific IgG antibody response. Furthermore, analysis of sera from IP and orally immunized animals revealed cross-reactive antibodies to numerous Salmonella isolates. Functional analysis of antibodies found within the sera from IP immunized animals revealed agglutination and opsonophagocytic activity against all tested O:4 Salmonella serovars. Together these results indicate that immunization with a S. Typhimurium wecA(-) strain confers protection against lethal challenge with wild type S. Typhimurium and S. Enteritidis and that immunization correlates with functional antibody production.

Characterization of Monoclonal Antibodies Which Recognize Specific Cell Surface Determinants on Salmonella Typhimurium

Murine typhoid, a naturally occurring disease in mice caused by Salmonella typhimurium, is a good experimental model with which to study the interactions between host and parasite and is especially important since the pathogenesis of this disease is similar to typhoid fever in man (1). Although the role of the immune system in the regulation of this disease has not been studied extensively, previous studies have suggested that the murine immune response to S. typhimurium was primarily regulated by T cells and macrophages (2). However, recent observations suggest that B cells, hence protective antibodies, also play an important role in resistance (3–5). Nevertheless, the identification of the salmonella cell surface antigens towards which these protective antibodies are directed remains controversial. One approach to the identification of these antigenic determinants would be to generate large quantities of antibody directed against a specific bacterial cell surface determinant and to use these antibodies in passive transfer experiments to determine their role in the protection of the host from murine typhoid.