The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress

A Francisella tularensis Schu S4 purine auxotroph is highly attenuated in mice but offers limited protection against homologous intranasal challenge.

Francisella tularensis is a gram-negative coccobacillus that causes the febrile illness tularemia. Subspecies that are pathogenic for humans include those comprising the type A (subspecies tularensis) or type B (subspecies holarctica) biovars. An attenuated live vaccine strain (LVS) developed from a type B isolate has previously been used to vaccinate at-risk individuals, but offers limited protection against high dose (>1000 CFUs) challenge with type A strains delivered by the respiratory route. Due to differences between type A and type B F. tularensis strains at the genetic level, it has been speculated that utilization of an attenuated type A strain as a live vaccine might offer better protection against homologous respiratory challenge compared with LVS. Here, we report the construction and characterization of an unmarked Delta purMCD mutant in the highly virulent type A strain Schu S4.Growth of Schu S4 Delta purMCD was severely attenuated in primary human peripheral blood monocyte-derived macrophages and in the A549 human lung epithelial cell line. The Schu S4 Delta purMCD mutant was also highly attenuated in mice when delivered via either the intranasal or intradermal infection route. Mice vaccinated intranasally with Schu S4 Delta purMCD were well protected against high dose intradermal challenge with virulent type A or type B strains of F. tularensis. However, intranasal vaccination with Schu S4 Delta purMCD induced tissue damage in the lungs, and conferred only limited protection against high dose Schu S4 challenge delivered by the same route. The level of protection observed was similar to that conferred following vaccination with wild-type LVS or the analogous LVS Delta purMCD mutant.Collectively, these results argue that development of the next generation live attenuated vaccine for Francisella should be based on use of the less pathogenic type B biovar rather than the more reactogenic type A biovar

Survival of mice vaccinated with Schu S4 Δ<i>purMCD</i> and challenged i.n. with 500 CFUs of wild-type Schu S4.

<p>Mice (n = 5) were vaccinated i.d. (A) or i.n. (B) with 10-fold serial dilutions of Schu S4 Δ<i>purMCD</i> ranging from 10 to 10⁶ CFUs. 21 days post-vaccination, mice were challenged i.n. with 500 CFUs of wild-type Schu S4. The survival of mice was then monitored for 14 days. Vaccination dose is listed in CFUs, and mean time-to-death was calculated for each dose. Asterisks indicate vaccinating doses at which MTD was significant compared to unvaccinated mice (*<i>P</i><0.05, **<i>P</i><0.001 by log-rank analysis).</p

Virulence of Schu S4 derivatives in BALB/c mice.

a<p>Survival, LD₅₀, and mean time-to-death (MTD) were determined 21 days after infection for the lowest Schu S4 and Schu S4 Δ<i>purMCD</i>∶pTZ752 doses (10 CFU) given, and the highest Schu S4 Δ<i>purMCD</i> dose (10⁶ CFU).</p>b<p>LD₅₀ values were calculated as described by Reed and Muench.</p

Growth of <i>F. tularensis</i> derivatives in mammalian cells.

<p>Intracellular growth of <i>F. tularensis</i> Schu S4 and LVS derivatives in human monocyte-derived macrophages (A and B), and A549 lung epithelial cells (C and D). Cells were infected with wild type <i>F. tularensis</i> LVS or Schu S4, LVS or Schu S4 Δ<i>purMCD</i>, or the LVS or Schu S4 genetically complemented Δ<i>purMCD</i>, and intracellular growth was monitored by lysing cells at the indicated time points and determining CFU by standard plate count. Infections were conducted at an MOI of 1, and gentamicin (5 µg/ml) was added to culture medium after infection to kill extracellular organisms. Standard errors of organ burden from three individual mice are shown. Asterisks indicate significance between wild-type or complemented Δ<i>purMCD</i> mutants and Δ<i>purMCD</i> burdens (<i>P</i><0.05; two-way ANOVA).</p

Vaccination and Challenge results.

a<p>MTD, mean time-to-death was calculated for a 21-day period.</p

Growth characteristics of Schu S4 derivatives in BALB/c mice.

<p>Groups of mice were infected i.n. (A, B, and C) or i.d. (D, E, and F) with wild-type Schu S4 (50 CFU), or one of two doses of Schu S4 Δ<i>purMCD</i> (10⁴ or 10⁶ CFU). At specific times after infection, subsets of mice were sacrificed and the numbers of bacteria present in the lungs (A and D), livers (B and E), or spleens (C and F) were determined by plating organ homogenates on MH agar containing ampicillin (50 µg/ml). Values represent the mean log CFU per ml from groups of three animals each. The horizontal dashed line represents the confidence level of detection for the assay. Standard errors of organ burdens for three individual mice are shown. Crosses indicate time points where n = 1; n = 3 for all other time points.</p