Identification of New Mycobacterium bovis antigens and development of a multiplexed serological bead-immunoassay for the diagnosis of bovine tuberculosis in cattle.

Serological assays for bovine tuberculosis diagnosis require the use of multiple Mycobacterium bovis specific antigens to ensure the detection of infected animals. In the present study, identification and selection process of antigens, based on data from published proteomic studies and involving the use of bioinformatics tools and an immuno-screening step, was firstly performed for identifying novel antigens that elicit an antibody response in M. bovis infection. Based on this approach, a panel of 10 M. bovis antigens [with known relevance (MPB70, MPB83, MPB70/83, and ESAT6/CFP10) and novel (Mb1961c, Mb1301c, Mb3871, Mb1403, Mb0592, and PE25/PPE41)] were constructed and thenused to develop a new multiplexed serological assay based on Luminex technology. The performance of the Luminex-bTB immunoassay was evaluated using sera from cattle with known tuberculosis status. Among the proteins whose ability to detect bovine tuberculosis was evaluated for the first time, PE25/PPE41 and Mb1403, but not Mb3871, showed good detection capacity. Following multiple antigen combination, the final Luminex-bTB immunoassay included seven antigens (MPB70, MPB83, MPB70/83, ESAT6/CFP10, PE25/PPE41, Mb1403, and Mb0592) and showed better global performance than the immunoassay using the four usual antigens (MPB70, MPB70/83, MPB83 and ESAT6/CFP10). The specificity and sensitivity values were, respectively, of 97.6% and 42.8% when the cut-off of two-positive antigens was used to classify samples as positive. With the use of the more-restrictive criterion of three-positive antigens, the specificity increased to 99.2% but the sensitivity decreased to 23.9%. The analysis of antigen profiles generated with the Luminex-bTB immunoassay showed that mainly serodominant proteins were recognized in samples from infected cattle. The detection of Mb1961c and Mb1301c appeared to be associated with presumed false-positive results. Moreover, sera from cattle originating from bTB-outbreaks but having inconclusive or negative skin test results were identified as positive by the Luminex-bTB immunoassay and showed an antigen pattern associated with M. bovis infection. The Luminex-bTB immunoassay including seven antigens may be useful as adjunct test for the detection of M. bovis-infected herds, and different cut-offs could be applied according to the bovine tuberculosis epidemiological context.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli.

Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.IMPORTANCEEscherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75-Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b.

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ.

Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, PG synthesis localizes at midcell under the control of a multiprotein complex, the divisome, allowing the safe formation of two new cell poles and separation of daughter cells. Genetic studies in Escherichia coli pointed out that FtsBLQ and FtsN participate in the regulation of septal PG (sPG) synthesis; however, the underlying molecular mechanisms remained largely unknown. Here we show that FtsBLQ subcomplex directly interacts with the PG synthase PBP1b and with the subcomplex FtsW-PBP3, mainly via FtsW. Strikingly, we discovered that FtsBLQ inhibits the glycosyltransferase activity of PBP1b and that this inhibition was antagonized by the PBP1b activators FtsN and LpoB. The same results were obtained in the presence of FtsW-PBP3. Moreover, using a simple thioester substrate (S2d), we showed that FtsBLQ also inhibits the transpeptidase domain of PBP3 but not of PBP1b. As the glycosyltransferase and transpeptidase activities of PBP1b are coupled and PBP3 activity requires nascent PG substrate, the results suggest that PBP1b inhibition by FtsBLQ will block sPG synthesis by these enzymes, thus maintaining cell division as repressed until the maturation of the divisome is signaled by the accumulation of FtsN, which triggers sPG synthesis and the initiation of cell constriction. These results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which seems to counterbalance the inhibitory effect of FtsBLQ to restore PBP1b activity.IMPORTANCE Bacterial cell division is governed by a multiprotein complex called divisome, which facilitates a precise cell wall synthesis at midcell and daughter cell separation. Protein-protein interactions and activity studies using different combinations of the septum synthesis core of the divisome revealed that the glycosyltransferase activity of PBP1b is repressed by FtsBLQ and that the presence of FtsN or LpoB suppresses this inhibition. Moreover, FtsBLQ also inhibits the PBP3 activity on a thioester substrate. These results provide enzymatic evidence of the regulation of the peptidoglycan synthase PBP1b and PBP3 within the divisome. The results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which functions to relieve the repression on PBP1b by FtsBLQ and to initiate septal peptidoglycan synthesis.