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A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.

ABSTRACT: Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The DeltapdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the DeltapdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the DeltapdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

SUBMITTER: Schmerk CL

PROVIDER: S-EPMC2889414 | biostudies-other | 2009 May

REPOSITORIES: biostudies-other

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A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.

Schmerk Crystal L CL Duplantis Barry N BN Howard Perry L PL Nano Francis E FE

Microbiology (Reading, England) 20090416 Pt 5

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The DeltapdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the l ...[more]

PMID: 19372155

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Project description:Francisella tularensis is the causative agent of tularemia and has gained recent interest as it poses a significant biothreat risk. F. novicida is commonly used as a laboratory surrogate for tularemia research due to genetic similarity and susceptibility of mice to infection. Currently, there is no FDA-approved tularemia vaccine, and identifying therapeutic targets remains a critical gap in strategies for combating this pathogen. Here, we investigate the soluble lytic transglycosylase or Slt in F. novicida, which belongs to a class of peptidoglycan-modifying enzymes known to be involved in cell division. We assess the role of Slt in biology and virulence of the organism as well as the vaccine potential of the slt mutant. We show that the F. novicida slt mutant has a significant growth defect in acidic pH conditions. Further microscopic analysis revealed significantly altered cell morphology compared to wild-type, including larger cell size, extensive membrane protrusions, and cell clumping and fusion, which was partially restored by growth in neutral pH or genetic complementation. Viability of the mutant was also significantly decreased during growth in acidic medium, but not at neutral pH. Furthermore, the slt mutant exhibited significant attenuation in a murine model of intranasal infection and virulence could be restored by genetic complementation. Moreover, we could protect mice using the slt mutant as a live vaccine strain against challenge with the parent strain; however, we were not able to protect against challenge with the fully virulent F. tularensis Schu S4 strain. These studies demonstrate a critical role for the Slt enzyme in maintaining proper cell division and morphology in acidic conditions, as well as replication and virulence in vivo. Our results suggest that although the current vaccination strategy with F. novicida slt mutant would not protect against Schu S4 challenges, the Slt enzyme could be an ideal target for future therapeutic development.

Project description:The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis, a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis-live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo, corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis, mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection.

Dataset Information

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.

Publications

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.

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