Putative Peptidoglycan-binding Domain Research Articles

Crystal structure of Type IX secretion system PorE C-terminal domain from Porphyromonas gingivalis in complex with a peptidoglycan fragment

Porphyromonas gingivalis, the major human pathogen associated to periodontal diseases, utilizes the Bacteroidetes-specific type IX secretion system (T9SS) to export virulence factors. PorE is a periplasmic multi-domain lipoprotein associated to the outer membrane that was recently identified as essential for T9SS function. Little is known on T9SS at the structural level, and in particular its interaction with peptidoglycan. This prompted us to carry out structural studies on PorE full length as well as on its four isolated domains. Here we report the crystal structure of the C-terminal OmpA_C-like putative peptidoglycan-binding domain at 1.55 Å resolution. An electron density volume was identified in the protein cleft, making it possible to build a naturally-occurring peptidoglycan fragment. This result suggests that PorE interacts with peptidoglycan and that PorE could anchor T9SS to the cell wall.

Binding from Both Sides: TolR and Full-Length OmpA Bind and Maintain the Local Structure of the E. coli Cell Wall

We present a molecular modeling and simulation study of the E.coli cell envelope, with a particular focus on the role of TolR, a native protein of the E.coli inner membrane, in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed binds peptidoglycan largely through electrostatic interactions. Non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides, respectively, maintain the position of the cell wall even in the absence of Braun's lipoprotein. The charged residues that mediate the cell-wall interactions of TolR in our simulations are conserved across a number of species of gram-negative bacteria.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

The periplasmic HrpB1 protein from Xanthomonas spp. binds to peptidoglycan and to components of the type III secretion system.

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 from Escherichia coli. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system.

Characterization of the Periplasmic Region of PomB, a Na + -Driven Flagellar Stator Protein in Vibrio alginolyticus

The stator proteins PomA and PomB form a complex that couples Na(+) influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB(C)). To investigate the function of PomB(C), a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB(C) fragment consisting of residues 135 to 315 (PomB(C₅) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB(ΔL)) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na(+) influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB(ΔL) stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a "plug" segment is important for the control of Na(+) influx through the stator complex as well as for stator assembly.

An IgG-binding protein A homolog in Staphylococcus hyicus

Shotgun phage display was used to identify a homolog of the IgG-binding protein staphylococcal protein A in Staphylococcus hyicus type strain CCUG 15602/ATCC 11249. This bacterium is the causative agent of exudative epidermitis in pigs and can also cause mastitis in cattle. A protein with similar features as the originally identified protein A in Staphylococcus aureus was described; an YSIRK-type signal peptide, four IgG-binding domains, a putative peptidoglycan-binding domain, and a cell wall anchoring motif (LPXTG) was present. The highest degree of similarity was to a protein A homolog in Staphylococcus pseudintermedius. However, typical Xr polypeptide repeats present in the protein A of S. aureus and S. pseudintermedius could not be identified in the protein A of S. hyicus. The presence of the spa gene in ten porcine and eight bovine clinical isolates of S. hyicus was investigated by PCR. In all isolates, the spa gene could be detected but the amplicons were of two sizes. Sequence analysis of four selected PCR amplicons showed that only three IgG-binding domains were present in the protein A of clinical isolates generating a smaller spa fragment. The finding of spa in S. hyicus contributes to an increased understanding of potential virulence factors in this species.

ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila

Aeromonas hydrophila uses the type II secretion system (T2SS) to transport protein toxins across the outer membrane. The inner membrane complex ExeAB is required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan-binding domain (Pfam number PF01471) conserved in many peptidoglycan-related proteins is present in the periplasmic region of ExeA (P-ExeA). In this study, co-sedimentation analysis revealed that P-ExeA was able to bind to highly pure peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in native PAGE, gel filtration and cross-linking experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at 30 degrees C and above. These results provide evidence that the putative peptidoglycan-binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions facilitate the multimerization of ExeA, favouring a model in which the protein forms a multimeric structure on the peptidoglycan during the ExeAB-dependent assembly of the secretin multimer in the outer membrane.

Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB

Torque generation in the Salmonella flagellar motor is coupled to translocation of H(+) ions through the proton-conducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan-binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C-terminal periplasmic fragment of MotB (MotB(C)) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton-conducting channel. Drastic conformational changes in the N-terminal portion of MotB(C) are required both for PG binding and the proton channel activation.

Structure of the Bacteriophage φKZ Lytic Transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis.

Staphylococcus epidermidis biofilm formation on polymer surfaces is considered a major pathogenicity factor in foreign-body-associated infections. Previously, the 148 kDa autolysin AtlE from S. epidermidis, which is involved in the initial attachment of the cells to polymer surfaces and also binds to the extracellular matrix protein vitronectin, was characterized. Here, the characterization of a novel autolysin/adhesin (Aae) in S. epidermidis is described. Aae was identified as a 35 kDa surface-associated protein that has bacteriolytic activity and binds vitronectin. Its N-terminal amino acid sequence was determined and the respective gene, aae, was cloned. DNA-sequence analysis revealed that aae encodes a deduced protein of 324 amino acids with a predicted molecular mass of 35 kDa. Aae contains three repetitive sequences in its N-terminal portion. These repeats comprise features of a putative peptidoglycan binding domain (LysM domain) found in a number of enzymes involved in cell-wall metabolism and also in some adhesins. Expression of aae by Escherichia coli and subsequent analysis revealed that Aae possesses bacteriolytic activity and adhesive properties. The interaction of Aae with fibrinogen, fibronectin and vitronectin was found to be dose-dependent and saturable and to occur with high affinity, by using the real-time Biomolecular Interaction Analysis (BIA). Aae binds to the Aalpha- and Bbeta-chains of fibrinogen and to the 29 kDa N-terminal fragment of fibronectin. In conclusion, Aae is a surface-associated protein with bacteriolytic and adhesive properties representing a new member of the staphylococcal autolysin/adhesins potentially involved in colonization.

Identification of a novel gene, fimV, involved in twitching motility in Pseudomonas aeruginosa.

Transposon mutagenesis was used to identify a new locus required for twitching motility in Pseudomonas aeruginosa. Four Tn5-B21 mutants which lacked twitching motility and a fifth which exhibited impaired motility were found to map to the same KPN:I restriction fragment at approximately 40 min on the P. aeruginosa genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 2.8 kb ORF, which was termed fimV. The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and anomalous electrophoretic migration, indicating unusual primary or secondary structure. The P. aeruginosa genome also possesses a paralogue of fimV. Homologues of fimV were also found in the sequenced genomes of the other type-IV-fimbriated bacteria Neisseria gonorrhoeae, Neisseria meningitidis, Legionella pneumophila and Vibrio cholerae, but not in those of other bacteria which lack type IV fimbriae. A fimV homologue was also found in the genome sequence of Shewanella putrefaciens, along with many other homologues of type IV fimbrial genes, indicating that this bacterium is also likely to produce type IV fimbriae. Wild-type twitching motility was restored to fimV mutants by complementation in a dosage-dependent manner. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.