Homologs of the LapD-LapG c-di-GMP Effector System Control Biofilm Formation by Bordetella bronchiseptica

Abstract

Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica

The Bordetella bronchiseptica nic locus encodes a nicotinic acid degradation pathway and the 6‐hydroxynicotinate‐responsive regulator BpsR

“…These observations are consistent with reports noting the stimulatory effects of Na on B . bronchiseptica biofilm formation (Irie et al ., ; Sisti et al ., ; Ambrosis et al ., ). In a study analyzing the effects of high Na concentrations on B. pertussis and B .…”

“Cell surface associated LapA ofPseudomonas fluorescensis anchored inside its Type-1 Secretion TolC-like Pore”

“…Additionally, ORF analysis programs often overlook or misannotate these large and complex adhesins (5, 16, 17). To overcome the first limitation, we took advantage of the observation that the two proteins that control LapA localization, the c-di-GMP-receptor LapD and the LapD-regulated protease LapG, show high sequence similarity and functional conservation between microbes (3, 12, 18) and can be identified by their respective domain architectures (LapG, pfam06035; LapD, pfam16448). We utilized the NCBI conserved domain database (CDD) and genome database to identify bacterial species encoding lapDG homologs; ∼1300 such lapGD -encoding species spanning 120 genera were identified.…”

“…Each annotated genome was investigated for proteins containing hallmarks of LapA: an N-terminal LapG cleavage site and C-terminal RTX motifs. To accomplish this task, we developed an algorithm to recognize large proteins (>1000 aa) with RTX-motifs (Dx[L/I]x(4)GxDx[L/I]xGGx(3)D) and a canonical N-terminal dialanine LapG cleavage motif ([T/A/P]AA[G/V]). Although our approach is constrained to properly annotated LapA-like ORFs, we still identified over 500 putative LapG substrates in ∼50 genera throughout Proteobacteria, including Legionella and Vibrio species (Table S1).…”

“…Although bacteria initiate surface contact and biofilm formation through a variety of strategies, many microbes need cell surface-associated protein adhesins to bind a surface. The family of giant (>200 kDa) T1S repeats-in-toxin (RTX)-containing adhesins is known to be critical for biofilm formation or surface binding by a variety of organisms, including Pseudomonas , Bordetella , Legionella , Vibrio, Shewanella and Marinomonas (3–8).…”

Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor

“…Recent work by our lab and others have focused on a conserved signaling system comprising the Lap operon that controls cell adhesion and biofilm formation in gammaproteobacteria, including Pseudomonas fluorescens (Chatterjee et al, 2014; Navarro et al, 2011; Newell et al, 2009, 2011b), P. aeruginosa (Cooley et al, 2016 Rybtke et al, 2015), P. putida (Gjermansen et al, 2010), Bordetella bronchiseptica (Ambrosis et al, 2016), and Shewanella oneidensis (Zhou et al, 2015) (Figure 1A). At its center, the inner membrane protein LapD functions as a receptor with degenerate GGDEF and EAL domains, which together relay intracellular c-di-GMP concentrations to the periplasm.…”