Role of BapA type III effector in the intracellular lifecycle of Burkholderia pseudomallei / Choh Leang Chung.

Burkholderia pseudomallei is a Gram-negative bacterium that causes the fatal disease, melioidosis. Treatment of melioidosis is difficult as the bacterium is intrinsically resistant to multiple antibiotics and there is a risk of recurrence despite prolonged and adequate antimicrobial therapy. To facilitate the development of effective prevention and eradication strategies, there is a need to gain further molecular insights into the molecular pathogenesis and intracellular lifecycle of this pathogen. B. pseudomallei utilises the type III secretion systems (T3SSs) to translocate effector proteins (T3SEs) directly into host cell cytoplasm to establish an intracellular infection to subvert the host immune systems. Herein, the putative T3SE protein, BapA, was characterised using bioinformatic analyses, in vitro and in vivo infection models and basic protein analyses. Bioinformatic analyses predicted the presence of a T3SE translocation signal in the first 100 amino acid sequence at the N-terminal of BapA amino acid sequence, indicating with a high probability that BapA is a T3SE of B. pseudomallei. Based on secondary structure prediction, de novo protein structure prediction models and intrinsically unstructured region prediction, the amino acid sequence after 320th amino acid at the C-terminal of BapA was predicted to be intrinsically unfolded. This may confer mechanistic roles in BapA function(s). The functional roles of BapA in the pathogenesis of B. pseudomallei were experimentally dissected by utilising a bapA knockout strain (ΔbapA) derived from the parental strain, B. pseudomallei K96243. In the in vitro infection assays using human cell lines, ΔbapA was significantly attenuated in adherence and invasion efficiencies onto and into A549 human lung epithelial cells, respectively. Phagocytosis of ΔbapA by U937 human macrophages was significantly reduced. A substantial decrease in the intracellular replication rate of ΔbapA was also observed in the U937 cells but not in A549 cells. In addition, the capacity of ΔbapA for cell-to-cell spread was significantly reduced without any defects in its actin-based intracellular motility. Collectively, it is rational to speculate that BapA may have essential role(s) in host cell entry into the host cell cytoplasm from the extracellular environment or from an infected neighbouring cell via actin-rich protrusion. These entry steps are the key steps required for B. pseudomallei to initiate its intracellular lifecycle and infection. The E. coli-expressed, purified recombinant BapA protein was analysed using SDS-PAGE. The intrinsically unstructured nature of BapA was demonstrated by the aberrant mobility of recombinant BapA on the SDS-PAGE. Presence of these dynamic regions in BapA may have a role in forming macromolecular assemblies by confering great flexibility and capacity in protein-protein or protein-ligand interactions. Thus, the intrinsically unstructured regions were hypothesised to confer the capability for BapA to exert its function in the different stages of B. pseudomallei intracellular lifecycle, as demonstrated in the current study. In conclusion, intrinsically unstructured BapA performs important roles in the intracellular entry of B. pseudomallei. Since cellular entry is an essential phase in the intracellular lifecycle, BapA T3SE is important in the pathogenesis of B. pseudomallei

Experimental Persistent Infection of BALB/c Mice with Small-Colony Variants of Burkholderia pseudomallei Leads to Concurrent Upregulation of PD-1 on T Cells and Skewed Th1 and Th17 Responses.

Burkholderia pseudomallei (B. pseudomallei), the causative agent of melioidosis, is a deadly pathogen endemic across parts of tropical South East Asia and Northern Australia. B. pseudomallei can remain latent within the intracellular compartment of the host cell over prolonged periods of time, and cause persistent disease leading to treatment difficulties. Understanding the immunological mechanisms behind persistent infection can result in improved treatment strategies in clinical melioidosis.Ten-day LD50 was determined for the small-colony variant (SCV) and its parental wild-type (WT) via intranasal route in experimental BALB/c mice. Persistent B. pseudomallei infection was generated by administrating sub-lethal dose of the two strains based on previously determined LD50. After two months, peripheral blood mononuclear cells (PBMCs) and plasma were obtained to investigate host immune responses against persistent B. pseudomallei infection. Lungs, livers, and spleens were harvested and bacterial loads in these organs were determined.Based on the ten-day LD50, the SCV was ~20-fold less virulent than the WT. The SCV caused higher bacterial loads in spleens compared to its WT counterparts with persistent B. pseudomallei infection. We found that the CD4+ T-cell frequencies were decreased, and the expressions of PD-1, but not CTLA-4 were significantly increased on the CD4+ and CD8+ T cells of these mice. Notably, persistent infection with the SCV led to significantly higher levels of PD-1 than the WT B. pseudomallei. Plasma IFN-γ, IL-6, and IL-17A levels were elevated only in SCV-infected mice. In addition, skewed plasma Th1 and Th17 responses were observed in SCV-infected mice relative to WT-infected and uninfected mice.B. pseudomallei appears to upregulate the expression of PD-1 on T cells to evade host immune responses, which likely facilitates bacterial persistence in the host. SCVs cause distinct pathology and immune responses in the host as compared to WT B. pseudomallei

One step growth curve of phage C34.

<p>The infection cycle of phage C34 propagating on <i>B</i>. <i>pseudomallei</i> strain CMS. E, eclipse period (30 minutes); L, latent period(40 minutes); B, burst size of the phage (234).</p

Bacterial burdens in lung, spleen, and liver of mice.

<p>Control (○), mice treated with i.p. phage treatment (2 × 10⁸ PFU of phage C34), administered 24 hours before the infection (■) or 2 hours post-infection (▲). The numbers of viable bacteria (CFU) were enumerated from the organs of mice on day 1, 2 and 3 post-infection (A-C). On day 3, the bacterial burden of mice which received post-infection treatment was significantly lower than that of the control in spleen tissues only.</p

Bacterial count of <i>B</i>. <i>pseudomallei</i> over the course of 6 hours.

<p>Control (●), <i>B</i>. <i>pseudomallei</i> infected by different MOIs (■) and the corresponding phage titre of C34 (▲).The graph shows averages for three independent assays.</p

Survivability of infected A549 cells.

<p>The survivability of infected A549 cells which received pre-infection and pre + post-infection treatment was significantly higher than the control. Survivability of non-infected A549 cells was calculated as 100% and thus not shown in the figure. The results are the averages of three independent assays. Pre: Pre-infection treatment; HT: Pre-infection treatment using heat-killed phage; Post: Post-infection treatment; Pre+post: Pre-infection and Post-infection treatment.</p

Mortality of <i>B</i>. <i>pseudomallei</i> strain CMS-infected mice.

<p>Mice which received treatment 24 hours before the infection (■) or 2 hours post-infection (▲). Control mice without any treatment (○) were 100% moribund at day 11 while 33% of the mice (n = 5) in both of the treated groups survived at the end of 14 days.</p

Transmission electron microscopy image of phage C34.

<p>The phages show an icosahedral head with contractile tail, which is a typical morphology of the family Myoviridae. Magnification: x100000.</p

Correlation analysis between Th1/Th2/Th17 cytokines.

<p><i>P</i> values were calculated using Spearman’s Rank Order Coefficient. *<i>P</i><0.008, **<i>P</i><0.002, ***<i>P</i><0.0002 after Bonferroni correction for 6 comparisons.</p

Ten-day survival rate of BALB/c mice infected with WT and SCV <i>B</i>. <i>pseudomallei</i>.

<p>BALB/c mice were infected with different doses of (A) OB and (B) OS, and ten-day survival rate of mice was plotted. (C) Comparison of ten-day survival rate of mice using same log CFU of OB and OS, and <i>P</i> value was calculated using Log-Rank test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Data are representative from 2 independent experiments (A-C, n = 6 per bacterial dose).</p