MglA Regulates Francisella tularensis subsp. novicida ( Francisella novicida ) Response to Starvation and Oxidative Stress

Abstract

MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments.Francisella tularensis is the causative agent of tularemia, a zoonotic disease that is typically transmitted by inhalation, by the bite of an infected arthropod, or by the ingestion of contaminated water. F. tularensis subsp. tularensis, subsp. holarctica, and subsp. mediasiatica are pathogens of humans and various other mammals (12). F. tularensis subsp. novicida (F. novicida) is infectious for immunocompromised humans only (22). All F. tularensis subspecies and F. novicida cause severe disease in mice, though their lethal doses vary in accordance to their propensity to cause human disease (12). Human pathogenic F. tularensis are highly infectious: fewer than 10 bacteria inoculated by the respiratory route can cause the disease in humans and result in a high incidence of mortality if untreated (12). The mechanisms that cause high F. tularensis infectivity and virulence are unknown. One possible mechanism is the remarkable ability of F. tularensis to evade innate immune responses early in infection. In mice infected with F. tularensis, proinflammatory cytokines and chemokines are usually measured only after a significant systemic organ burden and inflammatory granulocyte infiltration occurs (4, 9, 11).

Adaptation of Francisella tularensis to the Mammalian Environment Is Governed by Cues Which Can Be Mimicked In Vitro

“…Cumulatively, these observations suggest that expression of the genes previously associated with growth in vivo is also a normal part of log-phase growth in vitro and that inhibition of this expression (either by growth in MHB, as shown here, or by mutation of MglA [18]) has detrimental effects on bacterial growth/physiology. BHI-grown F. tularensis cells most closely resemble extracellular M-grown bacteria.…”

“…For IglB, the signal for M-grown bacteria was compared to that for BHI-grown cells. (2,5,8,18,24). To determine if growth in BHI was stimulating any MglAindependent changes, we also examined the phenotype(s) of an mglA mutant in this medium.…”

RelA regulates virulence and intracellular survival of Francisella novicida

“…Stress-induced (p)ppGpp production in Helicobacter pylori is required for survival during acid exposure and aerobic shock (Mouery et al, 2006;Wells & Gaynor, 2006), conditions typically encountered during the course of infection. In Francisella, intracellular replication and virulence are dependent on MglA and SspA, which regulates the FPI (Guina et al, 2007;Nano & Schmerk, 2007;Charity et al, 2007). In this study, we have identified a global regulatory system that plays a role in F. novicida virulence, where a relA mutant had reduced survival in macrophage-like cells, and was attenuated more than 100-fold in a BALB/c mouse model.…”

Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

“…77,2009 CHARACTERIZATION OF FRANCISELLA TRANSPOSON MUTANTS 1333 carB mutants in MDM was not achieved unless uracil was added to the tissue culture medium at the time of infection or shortly thereafter. Because we also found that IglC levels are normal in mutant 9H5, which contains one intact and one disrupted copy of iglC, and this strain exhibits no apparent virulence defects, it is likely that uracil deficiency also alters the expression of other, as-yet-unidentified virulence genes outside the iglABCD operon or the FPI, perhaps via effects on key regulatory factors, such as MglA, SspA, or FevR (9,13,15,17,37,47). Our data also suggest that either sustained access to uracil or higher concentrations of this nutrient are required for intracellular replication, reminiscent of a study by Fortier et al which demonstrated that the elevation of phagosome pH by using NH 4 Cl or related agents ablates LVS growth in murine peritoneal macrophages and enhances phagocytic killing by preventing bacterial access to transferrin iron (31).…”