Ed T. Buurman

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

Mnt1p and Mnt2p of Candida albicans Are Partially Redundant α-1,2-Mannosyltransferases That Participate in O-Linked Mannosylation and Are Required for Adhesion and Virulence

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant α-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Δ and mnt1Δ single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Δmnt2Δ mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.

Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence.

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn2+ as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca2+ and Mn2+ ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn2+-dependent mannosyltransferases. The Capmr1Δ null mutant was viable in vitro and had no growth defect even on media containing low Ca2+/Mn2+ ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.

A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host-fungus interactions.

The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTΔ null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of β-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.

In Vitro Validation of Acetyltransferase Activity of GlmU as an Antibacterial Target in Haemophilus influenzae

Background: A search was initiated to identify inhibitors of the acetyltransferase domain of GlmU that could be exploited as starting points for new antimicrobials. Results: Sulfonamide inhibitors were identified that upon chemical modification displayed antimicrobial activity mediated via GlmU. Conclusion: Enzymatic inhibition of GlmU can lead to antimicrobial activity. Significance: For the first time, GlmU was validated as an antimicrobial target in vitro. GlmU is a bifunctional enzyme that is essential for bacterial growth, converting d-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZenecas compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against Gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against Gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.

MOLECULAR CLONING AND SEQUENCING OF A CHITIN SYNTHASE GENE (CHS2) OF PARACOCCIDIOIDES BRASILIENSIS

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino‐terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane‐spanning regions at the carboxyl terminus. The nucleotide sequence of CHS2 and its flanking regions has been submitted to GenBank under Accession Number Y09231.

Novel rapidly diversifiable antimicrobial RNA polymerase switch region inhibitors with confirmed mode of action in Haemophilus influenzae.

A series of inhibitors with a squaramide core was synthesized following its discovery in a high-throughput screen for novel inhibitors of a transcription-coupled translation assay using Escherichia coli S30 extracts. The inhibitors were inactive when the plasmid substrate was replaced with mRNA, suggesting they interfered with transcription. This was confirmed by their inhibition of purified E. coli RNA polymerase. The series had antimicrobial activity against efflux-negative strains of E. coli and Haemophilus influenzae. Like rifampin, the squaramides preferentially inhibited synthesis of RNA and protein over fatty acids, peptidoglycan, and DNA. However, squaramide-resistant mutants were not cross-resistant to rifampin. Nine different mutations were found in parts of rpoB or rpoC that together encode the so-called switch region of RNA polymerase. This is the binding site of the natural antibiotics myxopyronin, corallopyronin, and ripostatin and the drug fidaxomicin. Computational modeling using the X-ray crystal structure of the myxopyronin-bound RNA polymerase of Thermus thermophilus suggests a binding mode of these inhibitors that is consistent with the resistance mutations. The squaramides are the first reported non-natural-product-related, rapidly diversifiable antibacterial inhibitors acting via the switch region of RNA polymerase.

Selective Inhibitors of Bacterial t-RNA-(N(1)G37) Methyltransferase (TrmD) That Demonstrate Novel Ordering of the Lid Domain.

The tRNA-(N(1)G37) methyltransferase (TrmD) is essential for growth and highly conserved in both Gram-positive and Gram-negative bacterial pathogens. Additionally, TrmD is very distinct from its human orthologue TRM5 and thus is a suitable target for the design of novel antibacterials. Screening of a collection of compound fragments using Haemophilus influenzae TrmD identified inhibitory, fused thieno-pyrimidones that were competitive with S-adenosylmethionine (SAM), the physiological methyl donor substrate. Guided by X-ray cocrystal structures, fragment 1 was elaborated into a nanomolar inhibitor of a broad range of Gram-negative TrmD isozymes. These compounds demonstrated no activity against representative human SAM utilizing enzymes, PRMT1 and SET7/9. This is the first report of selective, nanomolar inhibitors of TrmD with demonstrated ability to order the TrmD lid in the absence of tRNA.

The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

Background: Phenylalanyl-tRNA synthetase inhibitors have been shown to be efficacious in animal models of infection. Results: Inhibitors occupy a newly identified hydrophobic auxiliary binding pocket. Conclusion: Compound binding in this pocket leads to high screening hit rates, resistance frequencies, and elevated plasma protein binding. Significance: New inhibitors may be identified by avoiding the auxiliary pocket. The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Illicit Transport via Dipeptide Transporter Dpp is Irrelevant to the Efficacy of Negamycin in Mouse Thigh Models of Escherichia coli Infection

Negamycin is a hydrophilic antimicrobial translation inhibitor that crosses the lipophilic inner membrane of Escherichia coli via at least two transport routes to reach its intracellular target. In a minimal salts medium, negamycins peptidic nature allows illicit entry via a high-affinity route by hijacking the Dpp dipeptide transporter. Transport via a second, low-affinity route is energetically driven by the membrane potential, seemingly without the direct involvement of a transport protein. In mouse thigh models of E. coli infection, no evidence for Dpp-mediated transport of negamycin was found. The implication is that for the design of new negamycin-based analogs, the physicochemical properties required for cell entry via the low-affinity route need to be retained to achieve clinical success in the treatment of infectious diseases. Furthermore, clinical resistance to such analogs due to mutations affecting their ribosomal target or transport is expected to be rare and similar to that of aminoglycosides.