Abstract
ABSTRACTMotility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. The motility structures type IV pili and flagella are also involved in sensing surface contact, which modulates pathogenicity. In Pseudomonas aeruginosa, the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies of Geobacter sulfurreducens and Dichelobacter nodosus implicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used transcriptome sequencing (RNA-seq) analysis to identify genes in addition to pilA with changes in expression in the absence of pilR. Among the genes identified were 10 genes whose transcription increased in the pilA mutant but decreased in the pilR mutant, despite both mutants lacking T4P and pilus-related phenotypes. The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription of fleSR, and thus many genes in the FleSR regulon. As a result, pilSR deletion mutants had defects in swimming motility that were independent of the loss of PilA. Together, these data suggest that in addition to controlling T4P expression, PilSR could have a broader role in the regulation of P. aeruginosa motility and surface sensing behaviors.
Highlights
Motility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times
In designing this experiment, we considered the following. (i) pilR mutants lack expression of PilA. (ii) Loss of PilA contributes to a decrease in intracellular levels of the messenger molecule cyclic AMP [28]. (iii) There are more than 200 genes in P. aeruginosa that are at least partially cAMP dependent, including Vfr, a cAMP-binding virulence factor regulator [28]
To separate genes that are affected by the loss of PilA that occurs in both pilA and pilR mutants from the genes that are truly regulated by PilR, we categorized genes as those whose expression was changed in only the pilA or pilR backgrounds, versus both backgrounds, compared to the wild-type (WT) PAK strain
Summary
Motility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. Among the genes identified were 10 genes whose transcription increased in the pilA mutant but decreased in the pilR mutant, despite both mutants lacking T4P and pilusrelated phenotypes The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. We provide evidence supporting a new role for PilSR in regulating flagellumdependent swimming motility in addition to pilus-dependent twitching motility Even though both pilA and pilR mutants lack PilA and pili, we identified sets of genes downregulated in the pilR mutant and upregulated in a pilA mutant as well as genes downregulated only in a pilR mutant, independent of pilus expression. The majority of the pilus fiber is made of hundreds to thousands of subunits of the major pilin protein, PilA [13], the expression of which could be energetically costly to the cell if not tightly controlled
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