sep413

Staphylococcus aureus persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe S. aureus WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized by wild-type S. aureus, mice colonized with the spa variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing S. aureus and diminish pathogen persistence.

IMPORTANCE Staphylococcus aureus persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.

Precautions recommended when working with versatile unnatural amino acid

Molecular Trojan horse tricks microbes into unleashing weapon of their own destruction

Fillipe L. R. do Carmo, Houem Rabah, Rodrigo D. De Oliveira Carvalho, Floriane Gaucher, Barbara F. Cordeiro, Sara H. da Silva, Yves Le Loir, Vasco Azevedo, Gwénaël Jan

Bacterial Transferase MraY, a Source of Inspiration towards New Antibiotics.

Curr Med Chem. 2018 Mar 29;:

Authors: Fer MJ, Corre LL, Pietrancosta N, Evrard-Todeschi N, Olatunji S, Bouhss A, Calvet-Vitale S, Gravier-Pelletier C

Abstract
The bacterial resistance to antibiotics constitutes more than ever a severe public health problem. The enzymes involved in bacterial peptidoglycan biosynthesis are pertinent targets for developing new antibiotics, notably the MraY transferase that is not targeted by any marketed drug. Many research groups are currently working on the study or the inhibition of this enzyme. After a concise overview of the role, mechanism and inhibition of MraY, the structure-activity relationships of 5'-triazole-containing aminoribosyluridine inhibitors we previously synthetized will be presented. The recently published MraY X-ray structures allowed us to achieve a molecular virtual high-throughput screening of commercial data bases and our in-house library resulting in the identification of promising compounds for the further development of new antibiotics.

PMID: 29600753 [PubMed - as supplied by publisher]

Synthesis of avibactam derivatives and activity on β-lactamases and peptidoglycan biosynthesis enzymes of mycobacteria.

Chemistry. 2018 Mar 30;:

Authors: Edoo Z, Iannazzo L, Compain F, Li de la Sierra Gallay I, van Tilbeurgh H, Fonvielle M, Bouchet F, Le Run E, Mainardi JL, Arthur M, Ethève-Quelquejeu M, Hugonnet JE

Abstract
There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus since their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, we report access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction. The amoxicillin-DBO combinations were active indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg/ml for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, we investigated the latter compounds as inhibitors of L,D-transpeptidases (LDTs), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of LDTs by S-carbamoylation indicating that optimization of DBOs for LDT inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

PMID: 29601108 [PubMed - as supplied by publisher]

Related Articles

Cystathionine β-lyase is involved in D-amino acid metabolism.

Biochem J. 2018 Mar 28;:

Authors: Miyamoto T, Katane M, Saitoh Y, Sekine M, Homma H

Abstract
Non-canonical D-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical D-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical D-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these D-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical D-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed D- and L-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in L-methionine metabolism and the production of D-amino acids, as well as D- and L-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

PMID: 29592871 [PubMed - as supplied by publisher]

Related Articles

Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology.

Nat Commun. 2018 Mar 28;9(1):1263

Authors: Turner RD, Mesnage S, Hobbs JK, Foster SJ

Abstract
Biopolymer composite cell walls maintain cell shape and resist forces in plants, fungi and bacteria. Peptidoglycan, a crucial antibiotic target and immunomodulator, performs this role in bacteria. The textbook structural model of peptidoglycan is a highly ordered, crystalline material. Here we use atomic force microscopy (AFM) to image individual glycan chains in peptidoglycan from Escherichia coli in unprecedented detail. We quantify and map the extent to which chains are oriented in a similar direction (orientational order), showing it is much less ordered than previously depicted. Combining AFM with size exclusion chromatography, we reveal glycan chains up to 200 nm long. We show that altered cell shape is associated with substantial changes in peptidoglycan biophysical properties. Glycans from E. coli in its normal rod shape are long and circumferentially oriented, but when a spheroid shape is induced (chemically or genetically) glycans become short and disordered.

PMID: 29593214 [PubMed - in process]

Related Articles

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD.

Sci Rep. 2018 Mar 28;8(1):5313

Authors: Leisico F, V Vieira D, Figueiredo TA, Silva M, Cabrita EJ, Sobral RG, Ludovice AM, Trincão J, Romão MJ, de Lencastre H, Santos-Silva T

Abstract
Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

PMID: 29593310 [PubMed - in process]

Peptidoglycan modification by the catalytic domain of Streptococcus pneumoniae OatA follows a ping-pong bi-bi mechanism of action.

Biochemistry. 2018 Mar 29;:

Authors: Sychantha D, Clarke AJ

Abstract
Streptococcus pneumoniae amongst other Gram-positive pathogens produces O-acetylated peptidoglycan using the enzyme OatA. This process occurs through the transfer of an acetyl group from a donor to the hydroxyl group of an acceptor sugar. While it has been established that this process involves the extracellular, catalytic domain of OatA ( SpOatAC), mechanistic insight is still unavailable. This study examined the enzymatic characteristics of SpOatAC-catalyzed reactions through analysis of both pre-steady and steady-state kinetics. Our findings clearly show that SpOatAC follows a ping-pong bi-bi mechanism of action involving a covalent acetyl-enzyme intermediate. The modified residue was verified to be the catalytic nucleophile, Ser438. The pH dependence of the enzyme kinetics revealed that a single ionizable group is involved consistent with the participation of a His residue. Single turn-over kinetics of esterase activity demonstrated that k2 ≫ k3, revealing that the rate-limiting step for the hydrolytic reaction was the breakdown of the acetyl-enzyme with a half-life of >1 min. The previous assignment of Asn491 as an oxyanion hole residue was also confirmed as its replacement with Ala resulted in a 50-fold decrease in catalytic efficiency relative wild-type SpOatAC. However, this loss of catalytic efficiency was mostly due to a large increase in KM, suggesting that Asn491 contributes more to substrate binding.

PMID: 29595955 [PubMed - as supplied by publisher]

Imaging Bacterial Cell Wall Biosynthesis.

Annu Rev Biochem. 2018 Mar 29;:

Authors: Radkov AD, Hsu YP, Booher G, VanNieuwenhze MS

Abstract
Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent D-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research. Expected final online publication date for the Annual Review of Biochemistry Volume 87 is June 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

PMID: 29596002 [PubMed - as supplied by publisher]

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis.

Nature. 2018 Mar 28;:

Authors: Sjodt M, Brock K, Dobihal G, Rohs PDA, Green AG, Hopf TA, Meeske AJ, Srisuknimit V, Kahne D, Walker S, Marks DS, Bernhardt TG, Rudner DZ, Kruse AC

Abstract
The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

PMID: 29590088 [PubMed - as supplied by publisher]

The wall that surrounds bacteria to shield them from external assaults has long been a tantalizing target for drug therapies. Indeed, some of modern medicine's most reliable antibiotics disarm harmful bacteria by disrupting the proteins that build their protective armor.

Related Articles

Host-Polarized Cell Growth in Animal Symbionts.

Curr Biol. 2018 04 02;28(7):1039-1051.e5

Authors: Pende N, Wang J, Weber PM, Verheul J, Kuru E, Rittmann SKR, Leisch N, VanNieuwenhze MS, Brun YV, den Blaauwen T, Bulgheresi S

Abstract
To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog.

PMID: 29576473 [PubMed - indexed for MEDLINE]

Related Articles

Design of a Short Thermally Stable α-Helix Embedded in a Macrocycle.

Chembiochem. 2018 05 04;19(9):902-906

Authors: Wu H, Acharyya A, Wu Y, Liu L, Jo H, Gai F, DeGrado WF

Abstract
Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and Tm ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

PMID: 29417711 [PubMed - indexed for MEDLINE]

A new technique could allow researchers to better understand bacteria-host interactions over the course of an infection.

Related Articles

Membrane Potential Is Required for MurJ Function.

J Am Chem Soc. 2018 04 04;140(13):4481-4484

Authors: Rubino FA, Kumar S, Ruiz N, Walker S, Kahne DE

Abstract
MurJ, the flippase that exports the bacterial cell wall monomer Lipid II to the periplasm, is a target for new antibiotics, which are desperately needed to treat Gram-negative infections. Quantitative methods to monitor MurJ activity are required to characterize inhibitors but are challenging to develop because the lipid-linked substrate is not chemically altered in a flippase reaction. Here we show that MurJ inhibition can be quantified by measuring the accumulation of intracellular Lipid II using a biotin-tagging strategy. We have exploited this assay to show that MurJ is inhibited in the presence of a compound that dissipates the membrane potential. By probing cysteine accessibility we have found that under this condition MurJ relaxes into an inactive, outward-facing conformation reminiscent of that targeted by the peptide antibiotic LysM. We conclude that membrane potential is required for MurJ function in E. coli, and we anticipate that the ability to accumulate this inactive conformation will lead to structures useful for inhibitor design.

PMID: 29558128 [PubMed - indexed for MEDLINE]

Related Articles

Effect of the lipid II sugar moiety on bacterial transglycosylase: the 4-hydroxy epimer of lipid II is a TGase inhibitor.

Chem Commun (Camb). 2017 Jan 05;53(4):771-774

Authors: Chen KT, Lin CK, Guo CW, Chang YF, Hu CM, Lin HH, Lai Y, Cheng TR, Cheng WC

Abstract
Lipid II analogues bearing major modifications on the second sugar (GlcNAc) were synthesized and evaluated for their substrate activity toward TGases. Unexpectedly, N-deacetyled lipid II decreased its activity dramatically, and the C4-axial OH lipid II became an inhibitor (IC50 = 8 μM) with an approximately 14-fold increase in binding affinity toward TGase (25 vs. 27).

PMID: 27999831 [PubMed - indexed for MEDLINE]

Crystal structure of an intramembranal phosphatase central to bacterial cell-wall peptidoglycan biosynthesis and lipid recycling.

Nat Commun. 2018 Mar 20;9(1):1159

Authors: Workman SD, Worrall LJ, Strynadka NCJ

Abstract
Undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane protein that recycles the lipid carrier essential to the ongoing biosynthesis of the bacterial cell wall. Individual building blocks of peptidoglycan are assembled in the cytoplasm on undecaprenyl phosphate (C55-P) before being flipped to the periplasmic face, where they are polymerized and transferred to the existing cell wall sacculus, resulting in the side product undecaprenyl pyrophosphate (C55-PP). Interruption of UppP's regeneration of C55-P from C55-PP leads to the buildup of cell wall intermediates and cell lysis. We present the crystal structure of UppP from Escherichia coli at 2.0 Å resolution, which reveals the mechanistic basis for intramembranal phosphatase action and substrate specificity using an inverted topology repeat. In addition, the observation of key structural motifs common to a variety of cross membrane transporters hints at a potential flippase function in the specific relocalization of the C55-P product back to the cytosolic space.

PMID: 29559664 [PubMed - in process]

WIRED Columnist Maryn McKenna on how online patient surveys are driving doctors to overprescribe antibiotics and driving up the number of drug-resistant infections.

María-Alexandra Cañas, María-José Fábrega, Rosa Giménez, Josefa Badia, Laura Baldomà

Hoppy beer is all the rage among craft brewers and beer lovers, and now UC Berkeley biologists have come up with a way to create these unique flavors and aromas without using hops.

Related Articles

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis.

Nat Commun. 2018 Mar 14;9(1):1078

Authors: El Ghachi M, Howe N, Huang CY, Olieric V, Warshamanage R, Touzé T, Weichert D, Stansfeld PJ, Wang M, Kerff F, Caffrey M

Abstract
As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.

PMID: 29540682 [PubMed - in process]

Related Articles

Discovery of nanomolar desmuramylpeptide agonists of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) possessing immunostimulatory properties.

J Med Chem. 2018 Mar 15;:

Authors: Gobec M, Tomašič T, Štimac A, Frkanec R, Trontelj J, Anderluh M, Mlinaric-Rascan I, Jakopin Ž

Abstract
Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.

PMID: 29543461 [PubMed - as supplied by publisher]

To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon.The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin.E. faecium 16-346 was resistant to rifampicin (MIC >4 mg/L), erythromycin (MIC >4 mg/L), tetracycline (MIC >16 mg/L) and vancomycin (MIC 8 mg/L), but susceptible to teicoplanin (MIC 0.5 mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5 kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin.This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium.

Related Articles

Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes.

ACS Infect Dis. 2018 06 08;4(6):1007-1018

Authors: Lee W, Do T, Zhang G, Kahne D, Meredith TC, Walker S

Abstract
Targeted modification of bacterial chromosomes is necessary to understand new drug targets, investigate virulence factors, elucidate cell physiology, and validate results of -omics-based approaches. For some bacteria, reverse genetics remains a major bottleneck to progress in research. Here, we describe a compound-centric strategy that combines new negative selection markers with known positive selection markers to achieve simple, efficient one-step genome engineering of bacterial chromosomes. The method was inspired by the observation that certain nonessential metabolic pathways contain essential late steps, suggesting that antibiotics targeting a late step can be used to select for the absence of genes that control flux into the pathway. Guided by this hypothesis, we have identified antibiotic/counterselectable markers to accelerate reverse engineering of two increasingly antibiotic-resistant pathogens, Staphylococcus aureus and Acinetobacter baumannii. For S. aureus, we used wall teichoic acid biosynthesis inhibitors to select for the absence of tarO and for A. baumannii, we used colistin to select for the absence of lpxC. We have obtained desired gene deletions, gene fusions, and promoter swaps in a single plating step with perfect efficiency. Our method can also be adapted to generate markerless deletions of genes using FLP recombinase. The tools described here will accelerate research on two important pathogens, and the concept we outline can be readily adapted to any organism for which a suitable target pathway can be identified.

PMID: 29534563 [PubMed - indexed for MEDLINE]

The transpeptidase PbpA and non-canonical transglycosylase RodA of Mycobacterium tuberculosis play important roles in regulating bacterial cell lengths.

J Biol Chem. 2018 Mar 12;:

Authors: Arora D, Chawla Y, Malakar B, Singh A, Nandicoori VK

Abstract
The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, crosslinked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The Mtb genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a non-classical transglycosylase RodA. We elucidate their roles in regulating in vitro growth and in vivo survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, while RodA is identified to be a member of an emerging class of non-canonical transglycosylases. Phosphorylation of RodA at T463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both, bacterial survival as well as formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that while redundant enzymes likely compensate for the absence of RodA or PbpA during in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host.

PMID: 29530985 [PubMed - as supplied by publisher]