is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. inoculation via abrasions and minor cuts by contaminated soil or water, as well as inhalation of aerosols (1, 2). Typical clinical manifestations include pneumonia and sepsis, with high mortality rates even after appropriate antibiotic treatment (1, 3). Various underlying diseases, such as diabetes mellitus, chronic renal failure, and chronic lung diseases, are proven risk factors for the acquisition of clinically manifest melioidosis (1). As a facultative intracellular pathogen, is able to invade host cells, escape from the endolysosome, survive within the cytosol, and to spread directly from cell to cell (4,C6). Numerous virulence factors are known to contribute to this intracellular lifestyle, which is crucial for full virulence of the pathogen (7,C9). Riociguat distributor harbors a remarkable number of various secretion systems, including three type III and six type VI secretion systems (T3SS and T6SS, respectively) (6, 10,C12). T6SS are widely distributed among Gram-negative bacteria and are considered to inject effector proteins into eukaryotic or bacterial cells (13,C17). The hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein G (VgrG) are hallmarks of all T6SS and secreted components of the T6SS apparatus itself (10, 18). The secretion of Hcp is considered to be a reliable indicator for a functional T6SS (18). A recent study analyzed the relevance of each single Hcp that is present in the various T6SS clusters of for virulence (10). The authors found that only deletion of Hcp from cluster 1 (Hcp1) led to a strong attenuation of BprC, encoded within cluster 3 of the T3SS in (gene, located within the T6SS1 cluster of to virulence. We also examined the role of for intracellular Riociguat distributor survival and induction of multinucleated giant cell (MNGC) and actin tail formation in mammalian cells. Using quantitative real-time PCR, we investigated the influence of on the expression of other T6SS1-associated components and regulators. Finally, we examined the influence of on Hcp1 secretion. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. and strains were grown on LB agar or in LB broth at 37C. If not stated otherwise, antibiotics were added at the following concentrations: 50 g/ml ampicillin (Ap), 37.5 g/ml kanamycin (Km), 3,000 g/ml zeocin, and 25 g/ml polymyxin B. Kmr gene-containing plasmids used for conjugation of strain E8 were selected with 1,000 g/ml Km. All experiments with were carried out in biosafety level 3 (BSL3) laboratories. TABLE 1 Plasmids and bacterial Riociguat distributor strains used in this study ORF; Kmr AprThis study????pCR2.1-TOPO-(reporter gene; optimized gene22????pEX-KM5-(base vector with fragment24????pUC18TminiTncontaining ORFThis studyStrains????complemented mutantE8 Riociguat distributor derivate, ORF Riociguat distributor disrupted by TnmutantE8 derivate, ORF deletedThis study????????complemented mutantmutant with chromosomal miniTn((ORF deletedThis study Open in a separate window aAbbreviations: DAP, 2,6-diaminopimelic acid; Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Gmr, gentamycin resistant; Kmr, kanamycin resistant; Tcr, tetracycline resistant; Zeor, zeocin resistant. Transposon mutagenesis. strain E8 was mutagenized with Tnlocus. Targeted mutagenesis. To construct markerless deletion mutants, the mutant was constructed as previously described (44). Up- and downstream PCR products of the and (and pEX-Km5-RHO3 by heat shock transformation and Rabbit Polyclonal to NR1I3 selected on LB plates containing 37.5 g/ml Km and 400 g/ml 2,6-diaminopimelic acid (DAP). Grown colonies were incubated overnight in LB broth containing 300 g/ml DAP and 37.5 g/ml Km in a rotating incubator. For conjugation, 100 l of E8 overnight culture was mixed with 200 to 1 1,200 l of pEX-KM5-or pEX-Km5-containing RHO3 overnight culture, and LB broth was added to a final volume of 1.5 ml. Bacterial suspensions were centrifuged for 1 min at 6,000 deletion (system as recently described (23). The forward primer BPSS1504for-HindIII and reverse primer BPSS1504rev-KpnI (Table 2) were used to amplify the open reading frame (ORF) of E8 by using genomic DNA as the template. Following digestion, this fragment was cloned into the pUC18T mini-TnDH5, and delivered into the mutant by four-parent mating using the donor strain DH5(pUC18T mini-Tn7T-Zeo-helper strains [HB101(pRK2013) and DH5 (pTNS3)] as recently described (23, 24). Selection was performed on 4% glycerol-containing LB agar plates with zeocin and polymyxin B. Successful insertion of into the recipient strain was verified by PCR. The mini-Tnelements were transposed to the Tnsite downstream of.