Dual input control: activation of the Bartonella henselae VirB/D4 type IV secretion system by the stringent sigma factor RpoH1 and the BatR/BatS two‐component system

Abstract

SummaryThe co-ordinated expression of virulence factors is a critical process for any bacterial pathogen to colonize its host. Here we investigated the mechanisms of niche adaptation of the zoonotic pathogen Bartonella henselae by combining genetic approaches and shotgun proteomics. We demonstrated that expression of the VirB/D4 type IV secretion system (T4SS) and its secreted effector proteins require the alternative sigma factor RpoH1, which levels are controlled by the stringent response (SR) components DksA and SpoT. The RpoH1-dependent activation requires an active BatR/BatS two-component system (TCS) while BatR expression is controlled by RpoH1 and the SR components. Deletion of spoT results in a strong attenuation of VirB/D4 T4SS expression whereas dksA, rpoH1 or batR deletion fully abolishes its activity. In contrast to their activating effect on the VirB/D4 T4SS, which is critical at the early stage of host infection, SpoT and DksA negatively regulate the Trw T4SS, which mediates host-specific erythrocyte infection at a later stage of the colonization process. Our findings support a model where the SR signalling and the physiological pH-induced BatR/BatS TCS conjointly control the spatiotemporal expression of B. henselae adaptation factors during host infection.

Gene Transfer Agent Promotes Evolvability within the Fittest Subpopulation of a Bacterial Pathogen

“…To characterize BaGTA behavior across time in a population of Bartonella cells, we constructed a reporter strain carrying a transcriptional gfp fusion to the phage-related tail collar gene (BH13960, renamed bgtC ) located at the BaGTA locus. We monitored single-cell BaGTA expression dynamics upon growth in medium M199/10% fetal calf serum (hereafter M199), a host cell-free system commonly used to study the temporal progression of the Bartonella virulence program (Québatte et al., 2013). Time-course analysis using flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that the gfp-bgtC reporter was heterogeneously expressed within a subpopulation of cells.…”

“…The spoT gene encodes for an Rhs guanosine-tetraphosphate synthase enzyme (ppGpp synthase). SpoT consists of an N-terminal synthetase domain that catalyzes the formation of the stringent response alarmone ppGpp (Québatte et al., 2013). The alarmone ppGpp functions by binding to target proteins, leading to bacterial cells shutting down active growth and entering a dormant state that promotes survival (Steinchen and Bange, 2016).…”

“…To validate our findings from the TnSeq analysis, we used a panel of individual Tn insertion mutants for a subset of the identified bgt genes (Québatte et al., 2013) as donor. Subsequent quantification of transduction efficiencies revealed that insertions within bgtEHIJ and brrS completely abrogates genetic exchange (frequency <10 −7.5 , Figure 3C) confirming the requirement of these genes for a functional BaGTA.…”

“…# BIO-25006Illumina sequencing chemistry version v4IlluminaHiSeq SBS Kit v4iProof DNA polymeraseBioradCat. # 72-5301 Experimental Models : Organisms / Strains Escherichia coli MG1655Lab stockN/A Escherichia coli MFDpirFerrières et al., 2010JKE201 Bartonella henselae ATCC 49882T Sm r Schulein and Dehio, 2002RSE247 Bartonella henselae ATCC 49882T Sm r -P virB - gfp Québatte et al., 2013MQB528 Bartonella henselae 49882T Sm r bgtC - gfp This workMQB1366 Bartonella henselae 49882T Sm r Δ bgtCD This workMQB759 Bartonella henselae 49882T Sm r Δ bgtCD BH15040:: himarI (Km r )This workMQB1439 Bartonella henselae ATCC 49882T Sm r -P virB - gfp Δ bgtCD This workMQB1028 Bartonella henselae 49882T Sm r bgtC - gfp carrying pMQ165.1This workMQB1633 Bartonella henselae ATCC 49882T Sm r -P virB - gfp x:: himarI ( Km r )(Pool)This workMQB1380 Bartonella henselae ATCC 49882T Sm r -P virB - gfp bepG :: himarI ( Km r )This workMQB1562 Bartonella henselae ATCC 49882T Sm r -P virB :: himarI : gfp ( Gm r )Québatte et al., 2013MQT222 Bartonella henselae ATCC 49882T Sm r P virB - gfp bgtE :: himarI ( Gm r )Québatte et al., 2013MQT226 Bartonella henselae ATCC 49882T Sm r P virB - gfp bepG :: himarI (Km r )This workMQB1562 Bartonella henselae ATCC 49882T Sm r P virB - gfp pstB ::himarI (Gm r )Québatte et al., 2013MQT269 Bartonella henselae ATCC 49882T Sm r P virB - gfp proY ::hima r I (Gm r )Québatte et al., 2013MQT215 Bartonella henselae ATCC 49882T Sm r P virB - gfp bgtA ::himarI (Gm r )Québatte et al., 2013MQT265 Bartonella henselae ATCC 49882T Sm r P virB - gfp bgtH ::himarI (Gm r )Québatte et al., 2013MQT260 Bartonella henselae ATCC 49882T Sm r P virB - gfp bgtI ::himarI (Gm r )Québatte et al., 2013MQT274 Bartonella henselae ATCC 49882T Sm r P virB - gfp bgtJ ::himarI (Gm r )…”

“…The himarI transposon cassette was constructed as follows: a synthetic DNA template carrying the illumine adapter linker sequence was re-amplified using primers prMQ1736 and prMQ1730. The gentamicin resistance cassette was amplified from pML001 (Québatte et al., 2013) using prMQ1735 and prMQ1735 with the high fidelity iProof DNA polymerase (Biorad). The Ptac/lac promoter was amplified from a derivative of pCD353 (Dehio et al., 1998) using prMQ1706a and prMQ1777.…”