The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

Abstract

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. KEYWORDS Xanthomonas, peptidoglycan, Slt70, type III secretion, lytic transglycosylase, effector proteins T he pathogenicity of many Gram-negative plant-and animal-pathogenic bacteria depends on a type III secretion (T3S) system, which translocates bacterial effector proteins into eukaryotic cells (1). Type III effector proteins manipulate host cellular pathways to the benefit of the pathogen and thus promote bacterial proliferation (2). T3S systems are highly complex nanomachines that span both bacterial membranes and are associated with a pilus-like appendage, which serves as a transport channel for secreted proteins (3). Protein translocation into eukaryotic cells depends on the channel-like T3S translocon, which inserts into the host plasma membrane (3, 4).T3S systems usually consist of 20 to 25 proteins, nine of which are conserved in plant-and animal-pathogenic bacteria and constitute the core elements of the secretion apparatus. These include ring structures in the bacterial inner membrane (IM) and outer membrane (OM), which are associated with a predicted periplasmic inner rod (3). The IM ring interacts with components of the export apparatus, which is assembled by members of the conserved Sct (secretion and cellular translocation) R, S, T, U, and V families of transmembrane proteins (3). The letters refer to the Ysc proteins of the T3S system from Yersinia spp. (5, 6). Components of the export apparatus insert into the IM and interact with cytoplasmic parts of the T3S system, including the predicted cytoplasmic (C) ring and the ATPase complex, which is presumably involved in T3S substrate

The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii

“…Accordingly, it was observed they are required for assembly of flagella and/or pili and in Caulobacter crescentus, Neisseria gonorrhoeae, and Rhodobacter sphaeroides (Viollier and Shapiro, 2003;Stohl et al, 2013;Herlihey et al, 2016), the type III secretion system in Xanthomonas campestris pv. Vesicatoria (Hausner et al, 2017) and the type VI secretion system in E. coli and A. baumannii (Weber et al, 2016;Santin and Cascales, 2017). Given these observations, it was not surprising to note that the ∆mltB mutant was devoid of pili at its cell surface (Fig.…”

“…Accordingly, it was observed they are required for assembly of flagella and/or pili and in Caulobacter crescentus , Neisseria gonorrhoeae , and Rhodobacter sphaeroides (Viollier and Shapiro, ; Stohl et al, ; Herlihey et al, ), the type III secretion system in Xanthomonas campestris pv. Vesicatoria (Hausner et al, ) and the type VI secretion system in E . coli and A .…”

HrpB7 from Xanthomonas campestris pv. vesicatoria is an essential component of the type III secretion system and shares features of HrpO/FliJ/YscO family members

“…HrpB7‐PhoA Δ2–120 and PhoA Δ2‐120 ‐HrpB7 both complemented the hrpB7 mutant phenotype, suggesting that the PhoA Δ2–120 fusion partner did not interfere with HrpB7 function (Figure 5a). When analysed in Xcv strain 85*Δ phoA , which lacks the native phoA gene (Hausner et al, ), no PhoA activity of both fusions was detectable, suggesting that the N‐ and C‐termini of HrpB7 are not located in the periplasm (Figure 5b). Given the absence of predicted transmembrane domains in HrpB7, we conclude that HrpB7 localises to the cytoplasmic side of the IM.…”

“…PhoA Δ2–120 fusions are indicated by asterisks. The fusion between the periplasmic predicted lytic transglycosylase HpaH and PhoA Δ2–120 was analysed as positive control (Hausner et al, ). ECW, Early Cal Wonder…”

“…PhoA Δ2-120 fusions are indicated by asterisks. The fusion between the periplasmic predicted lytic transglycosylase HpaH and PhoA Δ2-120 was analysed as positive control (Hausner et al, 2017). ECW, Early Cal Wonder 85*ΔhrpB7, suggesting that protein function was not completely abolished ( Figure S5; Table 1).…”