Research On The Biological Function Of H3-T6SS In Pseudomonas Aeruginosa

Bacteria mediate interactions with their surroundings by releasing a variety of proteins into the extracellular milieu.Bacteria have evolved at least seven specialized secretory systems to accomplish this task,each releasing a discrete set of proteins through complex and genetically divergent apparatuses.One such system is the type VI secretion system（T6SS）,which is a contact-dependent protein export pathway that delivers toxic effectors into target bacterial and eukaryotic cells.T6 SS is widely distributed in Gram-negative bacteria.Three separate T6 SSs called H1-,H2-,and H3-T6 SS have been discovered in Pseudomonas aeruginosa PAO1.Recent studies suggest that,in contrast to the H1-T6 SS that targets prokaryotic cells,H2-T6 SS and H3-T6 SS are involved in interactions with both prokaryotic and eukaryotic cells.However,the detailed functions of T6 SS components are still uncharacterized.The intracellular multiplication factor（IcmF）protein is conserved in type VI secretion systems（T6SS）of all different bacterial pathogens.Bioinformatic analysis revealed that IcmF3 in P.aeruginosa PAO1 is different from other IcmF homologues and may represent a new branch of these proteins with distinct functions.Herein,we have investigated the function of IcmF3 in this strain.We have shown that deletion of the icmF3 gene in P.aeruginosa PAO1 is associated with pleiotropic phenotypes.The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium.Surprisingly,this mutant is also defective for the production of pyoverdine,as well as defects in swimming motility and virulence in a C.elegans worm model.The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces.Additionally,expression of two phenazine biosynthetic loci is increased in the icmF3 mutant,leading to the overproduction of pyocyanin.Finally,the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin.And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene.The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P.aeruginosa PAO1.The pleiotropic effects observed upon icmF3 deletion also suggest that H3-T6 SS may play critical roles in both pathogenesis and environmental adaptation in P.aeruginosa PAO1.Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P.aeruginosa infection.Important facets of the innate immune system are to limit iron availability and synthesize highly toxic reactive oxygen species（ROS）to invading P.aeruginosa.Our studies have shown that H3-T6 SS loci play an important role in overcoming the host defense of limiting iron availability in P.aeruginosa PAO1.A bioinformatic analysis of the distributed situation of T6 SS effectors in Pseudomonas aeruginosa PAO1 genome predicts that PA2374 containing MORN₂ domains may represent previously undiscovered H3-T6 SS substrate.And then,our results establish that PA2374 is a substrate secreted by H3-T6 SS.However,mutations in H3-T6 SS structural genes can not completely abrogated the secretions of PA2374 and Hcp3,indicating the existence of other transporters for recognizing PA2374 and Hcp3.Bacterial two-hybrid and GST pull-down have revealed that Hcp3 or VgrG3 directly interact with PA0095 and indirect interaction is detectable between PA2374 and PA0095,indicating PA2374 and Hcp3 secretions may require PA0095,which encodes VgrG1 b whose secretion has been demonstrated to be T6 SSindependent.Subsequently,GST pull-down and ITC have revealed that PA2374 is capable of binding Pseudomonas quinolone signal（PQS）and directly interact with FptA or OprF.Furthermore,the H3-T6 SS loci are induced by disruption of iron homeostasis.And,PA2374 is required for P.aeruginosa PAO1 iron acquisition by mediating PQS-Fe acquisition through outer membrane protein FptA and OprF.This process may be dependent on outer membrane vesicles serving as a transport vehicle for PA2374 and PQS-Fe in the extracellular environment.Competitive infections have demonstrated that H3-T6 SS and PA2374 play an important role in overcoming the host defense of sequestration and scavenging of iron in P.aeruginosa PAO1.Our studies have also shown that H3-T6 SS loci play an important role in overcoming the host defense of generating highly toxic reactive oxygen species（ROS）in P.aeruginosa PAO1.LC-MS and β-galactosidase assays have shown that the repression of the production of PQS by H3-T6 SS loci in P.aeruginosa PAO1 is exhibited and relieved in response to iron availability.In turn,PQS differentially regulates P.aeruginosa H3-T6 SS structural genes and secretion substrate gene PA2374.PA2374 expression is suppressed by PQS.Conversely,H3-T6 SS structural genes are positively controlled by PQS.Further,H3-T6 SS is required for resistance to H2O2 stress but dispensable for defense against other oxidative stress in P.aeruginosa.H3-T6 SS is also critical in neutralizing ROS accumulated in P.aeruginosa under H2O2 stress conditions.Meanwhile,expression of H3-T6 SS in P.aeruginosa is activated by RpoS.In contrast,the expression of H3-T6 SS in P.aeruginosa was not detectably affected by the deletion of oxyR and H2O2 stress.Furthermore,quantitative proteomics has been applied to study differentially expressed proteins of P.aeruginosa PAO1 compared with clpV3 mutant.We find two proteins involved in PQS synthesis and many proteins which significantly contribute to the oxidative stress response are upregulated in clpV3 mutant.In clpV3 mutant,two iron storage proteins Dps and BfrA have been shown to be downregulated,and a ferredoxin NADP reductase（Fpr）which is required for the efficient release of iron stored in iron storage proteins has been shown to be upregulated.Further,PQSdeficient mutant and dps overexpression are both to rescue the sensitivity to H2O2 stress exhibited by H3-T6 SS mutants.Finally,we propose a mechanism for H3-T6SS-facilitated oxidative resistance in P.aeruginosa: The repression of the production of PQS by H3-T6 SS in P.aeruginosa results in the upregulation of two iron storage proteins Dps and BfrA and the downregulation of Fpr,and then the levels of intracellular unincorporated iron are suppressed,it thereby attenuates the Fenton reaction and the highly toxic ROS generation that would otherwise result.