This observed down-regulation of important virulence-related
genes is consistent with the noticed virulence defects in the cellular infection studies with D. discoideum and human macrophages as hosts. Table 2 Gene expression of selected Type III secretion genes in the typA mutant compared to that in wild type PA14 using RT-qPCR Gene Fold change in gene expression in the typA mutant relative to wild typea T3SS exsA −3.1 ± 0.5 pscC Angiogenesis inhibitor −2.3 ± 0.4 pscJ −3.5 ± 0.3 pscT −5.1 ± 0.3 pcrV −5.8 ± 0.6 Discussion In this study, we have shown that TypA is involved in virulence of P. aeruginosa by analyzing the consequences of a typA knock-out on phagocytic amoebae and human macrophages as well as the interaction with the nematode C. elegans. Moreover, TypA also contributes to resistance to different antibiotics as well as attachment and biofilm formation in P. aeruginosa. TypA is a highly conserved prokaryotic GTPase exhibiting structural homologies to translation factor GTPases
such as EF-G and LepA and is described to associate with the ribosomes under normal bacterial growth [15, LY2874455 clinical trial 31]. In enteropathogenic E. coli (EPEC), TypA co-ordinates the expression of key stress and virulence factors including flagella, Type III secretion system as well as the LEE and the espC pathogenicity islands [18, 32] by regulating gene expression of major regulators such
as Ler, which in turn controls these respective pathogenicity islands. Consequently, it has been suggested that TypA is on a relatively high level in the complex regulatory hierarchy of virulence regulation in this organism [18, 32]. In contrast, analysis in Mycobacterium tuberculosis revealed that TypA does not act as a virulence regulator in this human pathogen, ruling out a general involvement of this protein in virulence regulation in pathogenic bacteria [33]. However, our results demonstrate that TypA plays a role in the pathogenesis of P. aeruginosa. The typA knock-out mutant exhibited a significant Aurora Kinase virulence deficiency in both the amoebae infection model and the macrophage uptake studies. These defects were comparable to a pscC mutant with a disrupted Type III secretion system and consistent with the down-regulation of Type III secretion genes during host-pathogen interaction. The Type III secretion system of Gram-negative bacteria is an important factor of pathogenesis and is involved in manipulating eukaryotic cells by injecting effector proteins into the host [27] and impacts diretly on bacterial uptake by phagocytic cells [30]. In P. aeruginosa, this complex, needle-like machinery is encoded by 36 genes and an important factor for the survival during interaction with phagocytic amoebae or human macrophages, among others [5, 29, 30].