Mn2+ and bacterial pathogenesis.

Fe2+ has traditionally been considered the most important divalent cation involved in host-pathogen interactions. However, recent research indicates a previously unappreciated role for transition metal divalent cations other than Fe2+ during infection. Recent studies have identified an absolute requirement for Mn2+ in bacterial pathogens that are Fe2+-independent, indicating an important role for Mn2+ in pathogenesis. Potential roles for Mn2+ in pathogenesis include effects on the detoxification of reactive oxygen intermediates (ROIs), as a cofactor for enzymes involved in intermediary metabolism and signal transduction, and as a stimulus for virulence gene regulation. This review focuses on how these possible roles for Mn2+ may affect bacterial pathogenesis and the outcome of an infection.

Mutations in yhiT enable utilization of exogenous pyrimidine intermediates in Salmonella enterica serovar Typhimurium.

Mutants capable of utilizing the pyrimidine biosynthetic intermediates carbamoylaspartate and dihydroorotate for growth were derived from pyrimidine auxotrophs of Salmonella enterica serovar Typhimurium LT2. The gain-of-function phenotypes both resulted from mutations in a single gene, yhiT, the third gene of a putative four-gene operon, yhiVUTS, for which there is no homologous region in Escherichia coli. Notably, when a mutant yhiT allele was transferred to a pyrimidine-requiring E. coli strain, the transformant was then capable of using carbamoylaspartate or dihydrorotate as a pyrimidine source. The operon arrangement of the yhiVUTS genes was supported by genetic analyses and studies employing RT-PCR, coupled to the determination of the transcriptional start site using 5'-random amplification of cDNA ends (RACE). Computer-generated predictions indicated that YhiT is an integral membrane protein with 12 putative transmembrane domains typical of bacterial transport proteins. Competition experiments showed that mutant YhiT interacts with the C4-dicarboxylates succinate and malate, as well as the amino acids aspartate and asparagine. The native function of wild-type YhiT remains undetermined, but the collective results are consistent with a role as a general transporter of C4-dicarboxylates and other compounds with a similar basic structure.

Genetic profiling of dendritic cells exposed to live- or ultraviolet-irradiated Chlamydia muridarum reveals marked differences in CXC chemokine profiles.

Chlamydia trachomatis is a major cause of sexually transmitted disease worldwide for which an effective vaccine is being actively pursued. Current vaccine efforts will be aided by elucidating the interaction between Chlamydia and dendritic cells (DCs). Protective immunity appears to develop slowly following natural infection in humans, and early vaccine trials using inactivated C. trachomatis resulted in partial, short-lived protection with possible enhanced inflammatory pathology during re-infection. Thus, immunity following natural infection with live chlamydia may differ fundamentally from immune responses induced by immunization with inactivated chlamydia. We explored this conjecture by studying the response of DCs exposed to either viable or inactivated [ultraviolet (UV) -irradiated] chlamydia elementary bodies (EBs; designated as Live-EB and UV-EB, respectively) using Affymetrix GeneChip microarrays. Thirty-one immunologically characterized genes were differentially expressed by DCs following exposure to Live-EB or UV-EB, including two glutamic acid-leucine-arginine cysteine-X-cysteine (ELR CXC) neutrophil chemoattractant chemokines, Cxcl1 (KC), and Cxcl2 (MIP-2). Up-regulation of these genes by Live-EB as compared to UV-EB was verified by quantitative reverse transcription-polymerase chain reaction and increased chemokine secretion was confirmed by enzyme-linked immunosorbent assay both in vitro and in vivo. Immunofluorescence and fluorescence-activated cell sorter analysis of chlamydia-infected lung tissue confirmed that Live-EB but not UV-EB induced significant DC and neutrophil infiltration during infection. These observations demonstrate that the development of an antichlamydial immune response is dramatically influenced by chlamydial viability. This has implications as to why early inactivated chlamydial vaccines were ineffective and suggests that new vaccine design efforts may benefit from in vitro DC screening for ELR chemokine expression profiles.

A live and inactivated Chlamydia trachomatis mouse pneumonitis strain induces the maturation of dendritic cells that are phenotypically and immunologically distinct.

The intracellular bacterial pathogen Chlamydia trachomatis is a major cause of sexually transmitted disease worldwide. While protective immunity does appear to develop following natural chlamydial infection in humans, early vaccine trials using heat-killed C. trachomatis resulted in limited and transient protection with possible enhanced disease during follow-up. Thus, immunity following natural infection with live chlamydia may differ from immune responses induced by immunization with inactivated chlamydia. To study this differing immunology, we used murine bone marrow-derived dendritic cells (DC) to examine DC maturation and immune effector function induced by live and UV-irradiated C. trachomatis elementary bodies (live EBs and UV-EB, respectively). DC exposed to live EBs acquired a mature DC morphology; expressed high levels of major histocompatibility complex (MHC) class II, CD80, CD86, CD40, and ICAM-1; produced elevated amounts of interleukin-12 and tumor necrosis factor alpha; and were efficiently recognized by Chlamydia-specific CD4+ T cells. In contrast, UV-EB-pulsed DC expressed low levels of CD40 and CD86 but displayed high levels of MHC class II, ICAM-1, and CD80; secreted low levels of proinflammatory cytokines; and exhibited reduced recognition by Chlamydia-specific CD4+ T cells. Adoptive transfer of live EB-pulsed DC was more effective than that of UV-EB-pulsed DC at protecting mice against challenge with live C. trachomatis. The expression of DC maturation markers and immune protection induced by UV-EB could be significantly enhanced by costimulation of DC ex vivo with UV-EB and oligodeoxynucleotides containing cytosine phosphate guanosine; however, the level of protection was significantly less than that achieved by using DC pulsed ex vivo with viable EBs. Thus, exposure of DC to live EBs results in a mature DC phenotype which is able to promote protective immunity, while exposure to UV-EB generates a semimature DC phenotype with less protective potential. This result may explain in part the differences in protective immunity induced by natural infection and immunization with whole inactivated organisms and is relevant to rational chlamydia vaccine design strategies.

Delivery of dangerous goods: type III secretion in enteric pathogens.

Type III secretion systems (TTSSs) of Gram-negative pathogens are molecular syringes that inject bacterial virulence factors directly into host cells. These virulence factors manipulate host cell pathways to aid bacterial survival within the host. Four important enteric pathogens use TTSSs to colonize and persist within the intestinal environment. The following is a brief review of the way in which TTSSs and their effectors contribute to the pathogenic nature of the prototypic diarrheal pathogens Salmonella, Shigella, Yersinia and enteropathogenic Escherichia coli (EPEC).

Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar typhimurium in the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the Salmonella-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this Salmonella pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of sifA mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells.

Host-pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes.

Nramp1 (Natural resistance-associated macrophage protein-1; also known as Slc11a1) is a host resistance gene that provides protection against several intracellular pathogens, including Salmonella enterica serovar Typhimurium. Little is known about the dynamic interplay that occurs between mammalian host resistance determinants such as Nramp1 and pathogens during infection. To explore these interactions, we examined the effect of Nramp1 on expression of Salmonella typhimurium (STM) virulence factors. We demonstrate that Salmonella pathogenicity island 2 (SPI2) is essential for replication of STM in spleens of infected Nramp1(+/+) mice. Furthermore, the presence of Nramp1 in transfected cell lines and congenic knockout mice resulted in the up-regulation of STM SPI2-associated virulence genes critical for intramacrophage survival. This Nramp1-dependent up-regulation of SPI2 was mimicked in vitro by chelation of iron, demonstrating the iron-responsive nature of expression of STM SPI2-associated virulence genes. We propose that acquisition of SPI2 by S. enterica not only enabled this bacterium to become an effective intracellular pathogen but also allowed the bacterium to withstand the effects of macrophage defense mechanisms such as Nramp1 early in the evolution of its pathogenic character. These dynamic Nramp1-pathogen interactions may be essential for regulating the course of an infection. This study demonstrates the presence of a previously undescribed direct influence of a mammalian innate host resistance locus on a pathogen at the genetic level.

The Salmonella enterica serovar typhimurium divalent cation transport systems MntH and SitABCD are essential for virulence in an Nramp1G169 murine typhoid model.

Nramp1 is a transporter that pumps divalent cations from the vacuoles of phagocytic cells and is associated with the innate resistance of mice to diverse intracellular pathogens. We demonstrate that sitA and mntH, genes encoding high-affinity metal ion uptake systems in Salmonella enterica serovar Typhimurium, are upregulated when Salmonella is internalized by Nramp1-expressing macrophages and play an essential role in systemic infection of congenic Nramp1-expressing mice.