Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme

Burkinshaw, Brianne J.; Deng, Wanyin; Lameignère, Emilie; Wasney, Gregory A.; Zhu, Haizhong; Worrall, L.J.; Finlay, B. Brett; Strynadka, N.C.J.

doi:10.1074/jbc.m115.639013

Cited by 47 publications

(67 citation statements)

References 59 publications

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“…Interestingly, many such PG lyases or muramidases are encoded within T3SS gene clusters [117][118][119][120][121] and a recent report even indicated an interaction between a lyase and the injectisome inner rod [121]. Similarly, in the flagellum, the PG lyase FlgJ has been shown to assist in the formation of the inner rod with its N-terminus, while the C-terminus exhibits a muramidase function [122,123].…”

Section: What Determines the Number And Cellular Distribution Of T3ss?mentioning

confidence: 99%

Type III secretion systems: the bacterial flagellum and the injectisome

Diepold

Armitage

2015

Phil. Trans. R. Soc. B

183

175

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One contribution of 12 to a theme issue 'The bacterial cell envelope'. The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications.

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Section: What Determines the Number And Cellular Distribution Of T3ss?mentioning

confidence: 99%

Type III secretion systems: the bacterial flagellum and the injectisome

Diepold

Armitage

2015

Phil. Trans. R. Soc. B

183

175

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“…Indeed, the bacterial flagellum is evolutionarily related to this secretion apparatus as many proteins associated with both systems are conserved in sequence and function (44,45). With EtgA from enteropathogenic E. coli, its localization is fixed (46) and its activity enhanced (47) by physical association with the inner rod protein EscI. The observation that R. sphaeroides SltF's activity is both fixed and enhanced by one structural protein and inhibited by another is novel.…”

Section: Discussionmentioning

confidence: 92%

“…However, it is becoming apparent that LTs are even more prevalent within bacteria as homologs and paralogs appear to be produced for specific tasks, such as the SltF for flagellum insertion described in this study. Indeed, the interaction of specialized LTs with components of secretion apparatuses appears to be a common theme, as others, such as EtgA from the injectisome type III secretion system (46,47) and VirB from the type IV secretion system (53), have been described. Such apparent redundancy reflects their important role for cell growth and survival, thus making them an attractive new target for the discovery of novel antibiotics (23).…”

Section: Discussionmentioning

confidence: 99%

Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF

et al. 2016

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SltF was identified previously as an autolysin required for the assembly of flagella in the alphaproteobacteria, but the nature of its peptidoglycan lytic activity remained unknown. Sequence alignment analyses suggest that it could function as either a muramidase, lytic transglycosylase, or ␤-N-acetylglucosaminidase. Recombinant SltF from Rhodobacter sphaeroides was purified to apparent homogeneity, and it was demonstrated to function as a lytic transglycosylase based on enzymatic assays involving mass spectrometric analyses. Circular dichroism (CD) analysis determined that it is composed of 83.4% ␣-structure and 1.48% ␤-structure and thus is similar to family 1A lytic transglycosylases. However, alignment of apparent SltF homologs identified in the genome database defined a new subfamily of the family 1 lytic transglycosylases. SltF was demonstrated to be endo-acting, cleaving within chains of peptidoglycan, with optimal activity at pH 7.0. Its activity is modulated by two flagellar rod proteins, FlgB and FlgF: FlgB both stabilizes and stimulates SltF activity, while FlgF inhibits it. Invariant Glu57 was confirmed as the sole catalytic acid/base residue of SltF. IMPORTANCEThe bacterial flagellum is comprised of a basal body, hook, and helical filament, which are connected by a rod structure. With a diameter of approximately 4 nm, the rod is larger than the estimated pore size within the peptidoglycan sacculus, and hence its insertion requires the localized and controlled lysis of this essential cell wall component. In many beta-and gammaproteobacteria, this lysis is catalyzed by the ␤-N-acetylglucosaminidase domain of FlgJ. However, FlgJ of the alphaproteobacteria lacks this activity and instead it recruits a separate enzyme, SltF, for this purpose. In this study, we demonstrate that SltF functions as a newly identified class of lytic transglycosylases and that its autolytic activity is uniquely modulated by two rod proteins, FlgB and FlgF.T he photosynthetic bacterium Rhodobacter sphaeroides is motile through the use of a single, subpolar flagellum (1). This locomotive organelle is tightly regulated and comprised of approximately 25 different proteins arranged into three major substructures: a basal body, hook, and thin helical filament. The basal body spans the bacterial cell envelope and contains the motor, a flagellum-specific type III export apparatus and at least four ringlike structures which are all connected by a filamentous rod (2). The rod assembles into a proximal rod that lies between the MS ring and the cell wall, which is composed of nine subunits of FliE (3) and six subunits of FlgB, FlgC, and FlgF (4), as well as a distal rod that is composed of 26 subunits of FlgG (5).During flagellum assembly, extensive modifications need to occur to the peptidoglycan (PG) sacculus to accommodate the insertion of the secretion apparatus, as well as to stabilize the function of this system by acting as an assembly scaffold (6, 7). PG is a heteropolymer of glycan strands and peptide chains forming a rig...

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“…It was therefore proposed that HrpB1 facilitates the stable anchoring of the T3S system to peptidoglycan. The interaction of HpaH with HrpB1 is reminiscent of the previous finding that the LT EtgA from EPEC interacts with the putative inner rod protein EscI (31,53). The binding of LTs to periplasmic components of the T3S system might help to control LT activity and to ensure that the degradation of peptidoglycan is locally restricted.…”

Section: Discussionmentioning

confidence: 56%

“…HpaH contains a predicted catalytic glutamate residue at position 58 (E58), which is conserved in homologous LTs and was shown to be required for the activity of the LT EtgA from EPEC (16,31) (Fig. S1).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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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

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Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme

Cited by 47 publications

References 59 publications

Type III secretion systems: the bacterial flagellum and the injectisome

Type III secretion systems: the bacterial flagellum and the injectisome

Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF

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

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