Gram-positive Cell Wall Research Articles

The architecture of the Gram-positive bacterial cell wall.

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of whichis the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60nm in diameter), deep (up to 23nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope framesour understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.

Binding of an antimicrobial peptide to bacterial cells: Interaction with different species, strains and cellular components

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores (“carpet” mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.

Comparative Molecular Dynamics Simulations Provide Insight Into Antibiotic Interactions: A Case Study Using the Enzyme L,L-Diaminopimelate Aminotransferase (DapL).

The L,L-diaminopimelate aminotransferase (DapL) pathway, a recently discovered variant of the lysine biosynthetic pathway, is an attractive pipeline to identify targets for the development of novel antibiotic compounds. DapL is a homodimer that catalyzes the conversion of tetrahydrodipicolinate to L,L-diaminopimelate in a single transamination reaction. The penultimate and ultimate products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are key components of the Gram-negative and Gram-positive bacterial peptidoglycan cell wall. Humans are not able to synthesize lysine, and DapL has been identified in 13% of bacteria whose genomes have been sequenced and annotated to date, thus it is an attractive target for the development of narrow spectrum antibiotics through the prevention of both lysine biosynthesis and peptidoglycan crosslinking. To address the common lack of structural information when conducting compound screening experiments and provide support for the use of modeled structures, our analyses utilized inferred structures from related homologous enzymes. Using a comprehensive and comparative molecular dynamics (MD) software package—DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations) 2.0, we investigated the binding dynamics of four previously identified antagonistic ligands of DapL from Verrucomicrobium spinosum, a non-pathogenic relative of Chlamydia trachomatis. Here, we present putative docking positions of the four ligands and provide confirmatory comparative molecular dynamics simulations supporting the conformations. The simulations performed in this study can be applied to evaluate putative targets to predict compound effectiveness prior to in vivo and in vitro experimentation. Moreover, this approach has the potential to streamline the process of antibiotic development.

Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.

A facile one pot synthesis of novel pyrimidine derivatives of 1,5-benzodiazepines via domino reaction and their antibacterial evaluation

A new series of pyrimidine (8, 14, 18 and 23) embellished analogues of 1,5-benzodiazepines were synthesized by the one-pot domino approach using the catalyst DABCO (1,4-diazabicyclo[2.2.2]octane). For each compound synthesized, anti-microbial efficacy was determined using broth microdilution assay and half maximal inhibitory concentration (IC50). Furthermore, FESEM (Field emission scanning electron microscope) studies were also carried out to observe the effect of the structure of test compounds on the morphology of both Gram-positive (S. aureus) and Gram-negative (E. coli) cell walls. The leakage of nucleotides and their integral components from compromised bacterial cells was assessed by plotting the optical density (OD) with respect to time of exposure at 320 nm. Anti-bacterial studies revealed that compound 23 was most active against targeted bacterial species. Results of the antibacterial study indicated that all the test compounds possess significant antibacterial potential against targeted bacterial strains. Amongst all, in the FE-SEM study, compound 23 caused marked alteration in bacterial cell morphology and resulted in maximum leakage of cell nucleotides in bacterial strains as compared to controls. Further efforts are required to establish their efficacy as antibacterial agents in clinical management.

Evaluation of modified Zobell marine agar for differential isolation of histamine-forming bacteria from fresh fish

A new series of pyrimidine (8, 14, 18 and 23) embellished analogues of 1,5-benzodiazepines were synthesized by the one-pot domino approach using the catalyst DABCO (1,4-diazabicyclo[2.2.2]octane). For each compound synthesized, anti-microbial efficacy was determined using broth microdilution assay and half maximal inhibitory concentration (IC50). Furthermore, FESEM (Field emission scanning electron microscope) studies were also carried out to observe the effect of the structure of test compounds on the morphology of both Gram-positive (S. aureus) and Gram-negative (E. coli) cell walls. The leakage of nucleotides and their integral components from compromised bacterial cells was assessed by plotting the optical density (OD) with respect to time of exposure at 320 nm. Anti-bacterial studies revealed that compound 23 was most active against targeted bacterial species. Results of the antibacterial study indicated that all the test compounds possess significant antibacterial potential against targeted bacterial strains. Amongst all, in the FE-SEM study, compound 23 caused marked alteration in bacterial cell morphology and resulted in maximum leakage of cell nucleotides in bacterial strains as compared to controls. Further efforts are required to establish their efficacy as antibacterial agents in clinical management.

The Gram-Positive Bacterial Cell Wall.

The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.

Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus

The Gram-positive cell wall consists of peptidoglycan functionalized with anionic glycopolymers, such as wall teichoic acid and capsular polysaccharide (CP). How the different cell wall polymers are assembled in a coordinated fashion is not fully understood. Here, we reconstitute Staphylococcus aureus CP biosynthesis and elucidate its interplay with the cell wall biosynthetic machinery. We show that the CapAB tyrosine kinase complex controls multiple enzymatic checkpoints through reversible phosphorylation to modulate the consumption of essential precursors that are also used in peptidoglycan biosynthesis. In addition, the CapA1 activator protein interacts with and cleaves lipid-linked CP precursors, releasing the essential lipid carrier undecaprenyl-phosphate. We further provide biochemical evidence that the subsequent attachment of CP is achieved by LcpC, a member of the LytR-CpsA-Psr protein family, using the peptidoglycan precursor native lipid II as acceptor substrate. The Ser/Thr kinase PknB, which can sense cellular lipid II levels, negatively controls CP synthesis. Our work sheds light on the integration of CP biosynthesis into the multi-component Gram-positive cell wall.

The efficacy of zero valent iron-sand filtration on the reduction of Escherichia coli and Listeria monocytogenes in surface water for use in irrigation

The use of surface and recycled water sources for irrigation can reduce demand on critical groundwater resources. Treatment or mitigation may be necessary for the use of these alternative water sources in order to reduce risk associated with microbial pathogens present in the water. In this study, the efficacy of a zero-valent iron (ZVI) sand filter was assessed for the reduction of Listeria monocytogenes and Escherichia coli in surface water. Water recovered from an agricultural pond was inoculated with E. coli TVS353 and an environmental L. monocytogenes isolate at 7 Log10 CFU/mL and horizontally filtered over a six-month period through a PVC pipe filter, filled with 35%:65% (volume:volume) ZVI:sand or sand alone. Filtered water was used to irrigate lettuce and bacterial persistence on lettuce leaves was determined for 7 days post-irrigation. Both ZVI:sand-filtered water and sand-filtered water contained significantly (p < 0.005) lower levels of E. coli and L. monocytogenes compared to initial unfiltered inoculated water. Population reductions of E. coli and L. monocytogenes were comparable after sand filtration. However, ZVI:sand filtration resulted in significantly greater population reductions of L. monocytogenes (P < 0.05) compared to E. coli. Populations of E. coli on leaves of lettuce plants irrigated with ZVI:sand-filtered water were not significantly lower than populations on plants irrigated with sand-filtered irrigation water over the 7-day period. However, populations of L. monocytogenes on lettuce leaves irrigated with ZVI-treated water were significantly lower than counts on plants irrigated with sand-filtered irrigation water on days 3 and 4 post irrigation (p = 0.052 and p = 0.042 for days 3 and 4, respectively. The differences observed in reductions of L. monocytogenes and E. coli by ZVI filtration is due to the differing effect that ZVI disruption has on Gram-positive and Gram-negative cell walls and membranes. ZVI- sand filters show promising results as an inexpensive on-farm technology for the mitigation of enteric foodborne bacterial populations in pond water over a six-month period.

Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses in the lung during murine pneumococcal pneumonia

BackgroundStreptococcus pneumoniae is a major causative agent in community-acquired pneumonia and sepsis. Overwhelming lung inflammation during pneumococcal pneumonia may hamper lung function. Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (Btk), a key signaling protein controlling the activation of various immune cells, including macrophages and neutrophils. The aim of this study was to determine whether ibrutinib treatment ameliorates acute lung inflammation during pneumococcal pneumonia.MethodsMice were treated orally with ibrutinib and the effect on acute pulmonary inflammation elicited by the gram-positive bacterial cell wall component lipoteichoic acid (LTA) and during ceftriaxone-treated pneumococcal pneumonia was assessed.ResultsTreatment with ibrutinib prior to and after intranasal LTA instillation reduced alveolar macrophage activation, neutrophil influx, cytokine release and plasma leakage into the lung. Postponed treatment with ibrutinib supplementing antibiotic therapy during ongoing pneumococcal pneumonia did not impair bacterial killing in lung, blood and spleen. In this setting, ibrutinib reduced alveolar macrophage and systemic neutrophil activation and substantially diminished further monocyte and neutrophil influx in the lung. In vitro, ibrutinib inhibited macrophage TNF secretion and neutrophil activation upon LTA and pneumococcal stimulation.ConclusionsTaken together, these data indicate that the Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses during acute pulmonary inflammation evoked by LTA and antibiotic-treated pneumococcal pneumonia and suggest that ibrutinib has the potential to inhibit ongoing lung inflammation in an acute infectious setting.

Electrochemical Studies on the Binding between Surface-Tethered DNA Aptamers and Lysozyme

Lysozyme is an enzyme that is responsible for the catalysis of β - 1,4 bond breakage in peptidoglycan residues of Gram-positive bacterium cell walls between N-acetylmuramic acid and N-acetylglucosamine. Lysozyme is commonly applied in food industry and its elevated concentration might indicate a development of leukemia or kidney failure, hence it is necessary to elaborate fast and sensitive tools for its detection.Herein, we present the studies on the elaboration of electrochemical aptasensor for lysozyme determination. For that purpose thiolated aptamer probes were immobilized on gold electrode surface that was subsequently modified with 6-mercapto-1-hexanol as blocking agent. It was found that 2h incubation period is sufficient to form monolayers that efficiently bind to target analyte. Methylene blue at 2 μM concentration was chosen as redox indicator as it provided the highest biosensor response in comparison to other tested electroactive molecules. 5 min. incubation time enabled lysozyme detection within the range from 0.1 fM to 10 nM. The developed biosensor exhibited good selectivity toward target analyte as well as minor response toward a reference DNA aptamer specific for HER2 biomarker. The proposed sensor was also successfully used for discrimination of lysozyme in diluted serum samples.

Effect of Inflammatory Mediators Lipopolysaccharide and Lipoteichoic Acid on Iron Metabolism of Differentiated SH-SY5Y Cells Alters in the Presence of BV-2 Microglia.

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), the Gram-negative and the Gram-positive bacterial cell wall components are important mediators of neuroinflammation in sepsis. LPS and LTA are potent activators of microglial cells which induce the production of various pro-inflammatory cytokines. It has been demonstrated that disturbance of iron homeostasis of the brain is one of the underlying causes of neuronal cell death but the mechanisms contributing to this process are still questionable. In the present study, we established monocultures of differentiated SH-SY5Y cells and co-cultures of differentiated SH-SY5Y cells and BV-2 microglia as neuronal model systems to selectively examine the effect of inflammatory mediators LPS and LTA on iron homeostasis of SH-SY5Y cells both in mono- and co-cultures. We monitored the IL-6 and TNFα secretions of the treated cells and determined the mRNA and protein levels of iron importers (transferrin receptor-1 and divalent metal transporter-1), and iron storing genes (ferritin heavy chain and mitochondrial ferritin). Moreover, we examined the relation between hepcidin secretion and intracellular iron content. Our data revealed that LPS and LTA triggered distinct responses in SH-SY5Y cells by differently changing the expressions of iron uptake, as well as cytosolic and mitochondrial iron storage proteins. Moreover, they increased the total iron contents of the cells but at different rates. The presence of BV-2 microglial cells influenced the reactions of SH-SY5Y cells on both LPS and LTA treatments: iron uptake and iron storage, as well as the neuronal cytokine production have been modulated. Our results demonstrate that BV-2 cells alter the iron metabolism of SH-SY5Y cells, they contribute to the iron accumulation of SH-SY5Y cells by manipulating the effects of LTA and LPS proving that microglia are important regulators of neuronal iron metabolism at neuroinflammation.

Inactivation of the Monofunctional Peptidoglycan Glycosyltransferase SgtB Allows Staphylococcus aureus To Survive in the Absence of Lipoteichoic Acid.

The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureussgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly.IMPORTANCE The bacterial cell wall acts as a primary defense against environmental insults such as changes in osmolarity. It is also a vulnerable structure, as defects in its synthesis can lead to growth arrest or cell death. The important human pathogen Staphylococcus aureus has a typical Gram-positive cell wall, which consists of peptidoglycan and the anionic polymers LTA and wall teichoic acid. Several clinically relevant antibiotics inhibit the synthesis of peptidoglycan; therefore, it and teichoic acids are considered attractive targets for the development of new antimicrobials. We show that LTA is required for efficient peptidoglycan cross-linking in S. aureus and inactivation of a peptidoglycan glycosyltransferase can partially rescue this defect, together revealing an intimate link between peptidoglycan and LTA synthesis.

Quantification of Lipoteichoic Acid in Hemodialysis Patients With Central Venous Catheters.

Hemodialysis patients with central venous catheters (CVCs) have chronic systemic inflammation, the source of which may be related to intraluminal bacterial biofilm. There is currently no non-invasive method to adequately evaluate intraluminal biofilm. Lipoteichoic acid (LTA) is a Gram-positive bacterial cell wall component that is spontaneously shed. The purpose of this study was to determine whether LTA could be quantified in biological samples and to evaluate potential relationships to markers of inflammation. Heparin-locked catheter aspirate was drawn from both the arterial and venous ports of each CVC prior to dialysis initiation. Venous blood from the dialysis circuit was collected 30 min after dialysis initiation. LTA was quantified in aspirate and plasma. Key markers of inflammation (interleukin-6, and hepcidin) and endothelial dysfunction (soluble vascular endothelial cadherin) were also determined in plasma samples. Catheter aspirate and systemic blood samples were obtained from 40 hemodialysis patients. The median (range) duration of catheter use was 130 (20–1635) days. Unexpectedly, median (range) plasma LTA concentrations (ng/mL) were significantly higher than catheter aspirate LTA concentrations [3.93 (0.25–15) vs. 2.38 (0.1–8.1), respectively, p = 0.01] in the majority (70%) of patients. Area under the receiver operator characteristic (ROC) curve showed good potential prognostic value of catheter aspirate LTA predicting systemic LTA concentrations with an area under the curve of 0.815 (95% CI, 0.68–0.95). A significant correlation was found between LTA and serum ferritin (r = 0.32, p = 0.04), however, there were no significant correlations between LTA and the other inflammation biomarkers assessed. LTA is quantifiable in aspirate and plasma of hemodialysis patients with CVCs and warrants further investigation to determine potential clinical application to intraluminal biofilm evaluation.

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens.

ABSTRACTGroup 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Bibersteinia trehalosi, and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis.

Selective recovery of RNAs from bacterial pathogens after their internalization by human host cells.

Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life. Here we present an RNA extraction protocol to selectively enrich the lowest bacterial RNA fraction from a mixture of human and bacterial cells, using zirconium beads, with minimal RNA degradation. Zirconium beads have higher capacity to extract bacterial RNAs than glass beads after pathogen internalization. We optimized the beads size and composition for an optimal bacterial lysis and RNA extraction. The protocol was validated on two human cell lines, differentiated macrophages and osteoblasts, with either Gram-positive (Staphylococcus aureus) or -negative (Salmonella typhimurium) bacteria. Relative to other published protocols, yield of total RNA recovery was significantly improved, while host cell infection was performed with a lower bacterial inoculum. Within the host, bacterial RNA recovery yields were about six-fold lower than an RNA extraction from pure bacteria, but the quality of the RNA recovered was essentially similar. Bacterial RNA recovery was more efficient for S. aureus than for S. typhimurium, probably due to their higher protection by the Gram-positive cell walls during the early step of eukaryotic cell lysis. These purified bacterial RNAs allow subsequent genes expression studies in the course of host cell-bacteria interactions.

Molecular recognition strategy for detection and antimicrobial susceptibility testing of Staphylococcus aureus by utilizing teicoplanin and porcine IgG as indicator molecules

Timely and accurate anti-infective therapy significantly benefits treatment of various life-threatening bacterial infectious diseases. Unfortunately, owing to the lack of selectivity for recognizing bacterial species, most of the reported phenotypic antimicrobial susceptibility testing protocols suffer from time-consuming bacterial isolation and identification. In this work, a highly-selective molecular recognition-based sandwich fluorimetric assay was developed to rapidly detect Staphylococcus aureus (S. aureus), and furtherly to assess its susceptibility to antibiotics. Immobilized porcine IgG was utilized to capture S. aureus through the selective interaction between Fc fragment of porcine IgG and protein A in the cell wall of S. aureus. Fluorescein isothiocyanate-labeled teicoplanin was adopted as the signal tracer utilizing the binding behavior between teicoplanin and d-Ala-d-Ala moieties in the peptidoglycans of Gram-positive bacterial cell wall. S. aureus could be detected within a wide linear rang of 1.0 × 103 to 1.0 × 107 CFU mL−1. Subsequently, this protocol utilizing porcine IgG and teicoplanin as the indicator molecules was utilized to assess the antimicrobial susceptibility of S. aureus after the bacteria were treated with antibiotics. The minimum inhibitory concentrations of penicillin, cefoxitin, clindamycin, trimethoprim/sulfamethoxazole and erythromycin were estimated to be >0.25,<4, <0.25, <2/38 and <0.5 μg mL−1, respectively. The whole process for antimicrobial susceptibility testing can be completed within 4 h since it avoided extra time-consuming isolation and identification of bacterial species.

Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanisms for wall teichoic acid glycosylation

The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria.