Redox-Mediated Inactivation of the Transcriptional Repressor RcrR is Responsible for Uropathogenic Escherichia coli's Increased Resistance to Reactive Chlorine Species.

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as a critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and is associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non-stress and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis; however, their responses to and defenses from HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared with other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator RcrR. We identified RcrR as a HOCl-responsive transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the derepression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl and phagocytosis indicating that the HOCl-mediated induction of the regulon plays a major role for UPEC's HOCl resistance. IMPORTANCE How do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in noninvasive enteropathogenic E. coli. Our data suggest expression of the target gene rcrB is exclusively responsible for UPEC's increased HOCl tolerance in culture and contributes to UPEC's survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Meta-Analysis of Immune Induced Gene Expression Changes in Diverse Drosophila melanogaster Innate Immune Responses.

Organisms are commonly infected by a diverse array of pathogens and mount functionally distinct responses to each of these varied immune challenges. Host immune responses are characterized by the induction of gene expression, however, the extent to which expression changes are shared among responses to distinct pathogens is largely unknown. To examine this, we performed meta-analysis of gene expression data collected from Drosophila melanogaster following infection with a wide array of pathogens. We identified 62 genes that are significantly induced by infection. While many of these infection-induced genes encode known immune response factors, we also identified 21 genes that have not been previously associated with host immunity. Examination of the upstream flanking sequences of the infection-induced genes lead to the identification of two conserved enhancer sites. These sites correspond to conserved binding sites for GATA and nuclear factor κB (NFκB) family transcription factors and are associated with higher levels of transcript induction. We further identified 31 genes with predicted functions in metabolism and organismal development that are significantly downregulated following infection by diverse pathogens. Our study identifies conserved gene expression changes in Drosophila melanogaster following infection with varied pathogens, and transcription factor families that may regulate this immune induction.

Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster.

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. Here, we characterize the Drosophila tuSz1 mutant strain, which mounts an aberrant immune response against its own fat body. We demonstrate that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Our data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals.

The S1A protease family members CG10764 and CG4793 regulate cellular immunity in Drosophila.

In nature, Drosophila melanogaster larvae are infected by parasitoid wasps and mount a cellular immune response to this infection. Several conserved signaling pathways have been implicated in coordinating this response, however our understanding of the integration and regulation of these pathways is incomplete. Members of the S1A serine protease family have been previously linked to immune functions, and our findings suggest roles for two S1A family members, CG10764 and CG4793 in the cellular immune response to parasitoid infection.

Immune Cell Production Is Targeted by Parasitoid Wasp Virulence in a Drosophila-Parasitoid Wasp Interaction.

The interactions between Drosophila melanogaster and the parasitoid wasps that infect Drosophila species provide an important model for understanding host-parasite relationships. Following parasitoid infection, D. melanogaster larvae mount a response in which immune cells (hemocytes) form a capsule around the wasp egg, which then melanizes, leading to death of the parasitoid. Previous studies have found that host hemocyte load; the number of hemocytes available for the encapsulation response; and the production of lamellocytes, an infection induced hemocyte type, are major determinants of host resistance. Parasitoids have evolved various virulence mechanisms to overcome the immune response of the D. melanogaster host, including both active immune suppression by venom proteins and passive immune evasive mechanisms. We identified a previously undescribed parasitoid species, Asobara sp. AsDen, which utilizes an active virulence mechanism to infect D. melanogaster hosts. Asobara sp. AsDen infection inhibits host hemocyte expression of msn, a member of the JNK signaling pathway, which plays a role in lamellocyte production. Asobara sp. AsDen infection restricts the production of lamellocytes as assayed by hemocyte cell morphology and altered msn expression. Our findings suggest that Asobara sp. AsDen infection alters host signaling to suppress immunity.