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Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit.  Burkholderia pseudomallei , the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH ™  labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during  in vitro  growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed  B .  pseudomallei bapA ,  bapB  and  bapC  mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the  bapB  mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the  bapA ,  bapB  and  bapC  mutants showed a minor reduction of  in vivo  fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full  B .  pseudomallei in vivo  fitness.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE