The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo . The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

The opportunistic human pathogen Pseudomonas aeruginosa effectively colonizes host epithelia using pili as primary adhesins. Here we uncover a surfacespecific asymmetric virulence program that enhances P. aeruginosa host colonization. We show that when P. aeruginosa encounters surfaces, the concentration of the second messenger c-di-GMP increases within a few seconds. This leads to surface adherence and virulence induction by stimulating pili assembly through activation of the c-di-GMP receptor FimW. Surface-attached bacteria divide asymmetrically to generate a piliated, surface-committed progeny (striker) and a flagellated, motile offspring that leaves the surface to colonize distant sites (spreader). Cell differentiation is driven by a phosphodiesterase that asymmetrically positions to the flagellated pole, thereby maintaining c-di-GMP levels low in the motile offspring. Infection experiments demonstrate that cellular asymmetry strongly boosts infection spread and tissue damage. Thus, P. aeruginosa promotes surface colonization and infection transmission through a cooperative virulence program that we termed Touch-Seed-and-Go.

The ability of Ralstonia solanacearum to cause disease in plants depends on its type III secretion system (T3SS). The expression of the T3SS and its effector substrates is coordinately controlled by a regulatory cascade, at the bottom of which is HrpB. Transcription of the hrpB gene is activated by a plant-responsive regulator named HrpG, which is a master regulator of a wide array of pathogenicity functions in R. solanacearum. We have identified in the genome of strain GMI1000 a close paralog of hrpG (83% overall similarity at the protein level) that we have named prhG. Despite this high similarity, the expression pattern of prhG is remarkably different from that of hrpG: prhG expression is activated after growth of bacteria in minimal medium but not in the presence of host cells, while hrpG expression is specifically induced in response to plant cell signals. We provide genetic evidence that prhG is a transcriptional regulator that, like hrpG, controls the expression of hrpB and the hrpB-regulated genes under minimal medium conditions. However, the regulatory functions of prhG and hrpG are distinct: prhG has no influence on hrpB expression when the bacteria are in the presence of plant cells, and transcriptomic profiling analysis of a prhG mutant revealed that the PrhG and HrpG regulons have only one pathogenicity target in common, hrpB. Functional complementation experiments indicated that PrhG and HrpG are individually sufficient to activate hrpB expression in minimal medium. Rather surprisingly, a prhG disruption mutant had little impact on pathogenicity, which may indicate that prhG has a minor role in the activation of T3SS genes when R. solanacearum grows parasitically inside the plant. The cross talk between pathogenicity regulatory proteins and environmental signals described here denotes that an intricate network is at the basis of the bacterial disease program.

The α-proteobacterial genus Bartonella comprises a group of ubiquitous mammalian pathogens that are studied as a model for the evolution of bacterial pathogenesis. Vast abundance of two particular phylogenetic lineages of Bartonella had been linked to enhanced host adaptability enabled by lineage-specific acquisition of a VirB/D4 type IV secretion system (T4SS) and parallel evolution of complex effector repertoires. However, the limited availability of genome sequences from one of those lineages as well as other, remote branches of Bartonella has so far hampered comprehensive understanding of how the VirB/D4 T4SS and its effectors called Beps have shaped Bartonella evolution. Here, we report the discovery of a third repertoire of Beps associated with the VirB/D4 T4SS of B. ancashensis, a novel human pathogen that lacks any signs of host adaptability and is only distantly related to the two species-rich lineages encoding a VirB/D4 T4SS. Furthermore, sequencing of ten new Bartonella isolates from under-sampled lineages enabled combined in silico analyses and wet lab experiments that suggest several parallel layers of functional diversification during evolution of the three Bep repertoires from a single ancestral effector. Our analyses show that the Beps of B. ancashensis share many features with the two other repertoires, but may represent a more ancestral state that has not yet unleashed the adaptive potential of such an effector set. We anticipate that the effectors of B. ancashensis will enable future studies to dissect the evolutionary history of Bartonella effectors and help unraveling the evolutionary forces underlying bacterial host adaptation.