Chronic Murine Typhoid Fever Is a Natural Model of Secondary Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory clinical syndrome associated with neoplastic disorders especially lymphoma, autoimmune conditions, and infectious agents including bacteria, viruses, protozoa and fungi. In both human and veterinary medicine, hemophagocytic histiocytic disorders are clinically important and frequently fatal. HLH in humans can be a primary (familial, autosomal recessive) or secondary (acquired) condition, with both types generally precipitated by an infectious agent. Previously, no mouse model for secondary HLH has been reported. Using Salmonella enterica serotype Typhimurium by oral gavage to mimic naturally-occurring infection in Sv129S6 mice, we characterized the clinical, hematologic and morphologic host responses to disease thereby describing an animal model with the clinico-pathologic features of secondary HLH as set forth by the Histiocyte Society: fever, splenomegaly, cytopenias (anemia, thrombocytopenia), hemophagocytosis in bone marrow and spleen, hyperferritinemia, and hypofibrinogenemia. Disease severity correlates with high splenic and hepatic bacterial load, and we show disease course can be monitored and tracked in live animals. Whereby secondary HLH is known to occur in human patients with typhoid fever and other infectious diseases, our characterization of a viable natural disease model of secondary HLH offers an important means to elucidate pathogenesis of poorly understood mechanisms of secondary HLH and investigation of novel therapies. We characterize previously unreported secondary HLH in a chronic mouse model of typhoid fever, and novel changes in hematology including decreased tissue ferric iron storage that differs from classically described anemia of chronic disease. Our studies demonstrate S. Typhimurium infection of mice is a natural infectious disease model of secondary HLH that may have utility for elucidating disease pathogenesis and developing novel therapies

The Chaperone IpgC Copurifies with the Virulence Regulator MxiE▿

The expression of a subset of Shigella flexneri virulence genes is dependent upon a cytoplasmic chaperone, IpgC, and an activator from the AraC/XylS family, MxiE. In this paper, we report that the chaperone forms a specific and stable heteromer with MxiE

CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp . One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp ). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE . In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp :: lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC

Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin

A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC▿

Antimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs. Salmonella enterica deploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. The ydeI gene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here that ydeI is additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS). ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates that ompD contributes to polymyxin B resistance, and both ydeI and ompD are important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs

Clinico-pathologic features of HLH in <i>S.</i> Typhimurium-infected mice.

<p>PI indicates post-oral infection with 2.0×10⁹ CFU <i>Salmonella enterica</i> serotype Typhimurium; C indicates mock-infected control mice.</p>*<p><i>P</i><0.05, Student's <i>t</i>-Test.</p>†<p>Independent experiment, same bacterial dosing and range for splenic bacterial CFU results.</p>‡<p>Formal diagnostic criteria for HLH per the Histiocyte Society guidelines <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009441#pone.0009441-Henter1" target="_blank">[5]</a>.</p>§<p>Consistent with a diagnosis of HLH, and ^Π strong supportive evidence for HLH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009441#pone.0009441-Henter1" target="_blank">[5]</a>.</p

Hematology of <i>S.</i> Typhimurium-infected mice: acute, then chronic active inflammatory response; microcytic anemia, persistent microcytosis.

<p>Mice were orally gavaged with 9.1×10⁸ CFU of <i>S</i>. Typhimurium (n = 8) or sterile PBS (n = 7). Complete blood counts were monitored over 16 weeks. X  =  <i>S</i>. Typhimurium-infected mice; circle  =  mock-infected control mice. Mean and standard deviation are shown. (A) neutrophils, (B) monocytes, (C) lymphocytes, (D) hematocrit (HCT), (E) mean cell volume (MCV). *<i>P</i><0.05 (Student's <i>t</i>-test).</p