Draft whole-genome sequences of three Borrelia burgdorferi isolates from Western Canada.

Whole-genome sequences are presented for three Borrelia burgdorferi, a causative agent of Lyme disease in North America, isolated from Ixodes pacificus ticks collected in British Columbia, Canada. Shotgun DNA libraries were prepared with Illumina DNA Prep and sequenced using the MiniSeq platform. Genome assemblies enabled multilocus sequence typing and ospC typing.

Complete genome sequences of bacteria isolated from cockroaches collected in a Bangladeshi hospital.

We report the complete genome sequences of four bacterial strains that were isolated from Blattella germanica (German cockroaches) that were found in three wards of the Rajshahi Medical College Hospital. Multiple antibiotic resistance genes were identified in each genome, with one genome containing multiple plasmid-encoded resistance genes.

Emergence of Inducible Macrolide Resistance in Mycobacterium chelonae Due to Broad-Host-Range Plasmid and Chromosomal Variants of the Novel 23S rRNA Methylase Gene, erm(55).

Macrolides are a mainstay of therapy for infections due to nontuberculous mycobacteria (NTM). Among rapidly growing mycobacteria (RGM), inducible macrolide resistance is associated with four chromosomal 23S rRNA methylase (erm) genes. Beginning in 2018, we detected high-level inducible clarithromycin resistance (MICs of ≥16µg/mL) in clinical isolates of Mycobacterium chelonae, an RGM species not previously known to contain erm genes. Using whole-genome sequencing, we identified a novel plasmid-mediated erm gene. This gene, designated erm(55)P, exhibits <65% amino acid identity to previously described RGM erm genes. Two additional chromosomal erm(55) alleles, with sequence identities of 81% to 86% to erm(55)P, were also identified and designated erm(55)C and erm(55)T. The erm(55)T is part of a transposon. The erm(55)P allele variant is located on a putative 137-kb conjugative plasmid, pMchErm55. Evaluation of 133 consecutive isolates from 2020 to 2022 revealed 5 (3.8%) with erm(55). The erm(55)P gene was also identified in public data sets of two emerging pathogenic pigmented RGM species: Mycobacterium iranicum and Mycobacterium obuense, dating back to 2008. In both species, the gene appeared to be present on plasmids homologous to pMchErm55. Plasmid-mediated macrolide resistance, not described previously for any NTM species, appears to have spread to multiple RGM species. This has important implications for antimicrobial susceptibility guidelines and treatment of RGM infections. Further spread could present serious consequences for treatment of other macrolide-susceptible RGM. Additional studies are needed to determine the transmissibility of pMchErm55 and the distribution of erm(55) among other RGM species.

A Complete Whole-Genome Sequence of Ignavigranum ruoffiae Strain CPL 242382-20, an Opportunistic Human Pathogen Recovered from a Breast Cyst.

Ignavigranum ruoffiae is a rare human pathogen. Strain CPL 242382-20 was isolated in Manitoba, Canada, from a breast cyst. Whole-genome sequencing was performed with the Oxford Nanopore Technologies MinION and Illumina MiSeq platforms. The circular chromosome is 1,949,382 bp with 39.68% G+C content and 1,765 protein-coding genes.

Structural Insights into the Inhibition of Undecaprenyl Pyrophosphate Synthase from Gram-Positive Bacteria.

The polyprenyl lipid undecaprenyl phosphate (C55P) is the universal carrier lipid for the biosynthesis of bacterial cell wall polymers. C55P is synthesized in its pyrophosphate form by undecaprenyl pyrophosphate synthase (UppS), an essential cis-prenyltransferase that is an attractive target for antibiotic development. We previously identified a compound (MAC-0547630) that showed promise as a novel class of inhibitor and an ability to potentiate ß-lactam antibiotics. Here, we provide a structural model for MAC-0547630's inhibition of UppS and a structural rationale for its enhanced effect on UppS from Bacillus subtilis versus Staphylococcus aureus. We also describe the synthesis of a MAC-0547630 derivative (JPD447), show that it too can potentiate ß-lactam antibiotics, and provide a structural rationale for its improved potentiation. Finally, we present an improved structural model of clomiphene's inhibition of UppS. Taken together, our data provide a foundation for structure-guided drug design of more potent UppS inhibitors in the future.

CryoEM structure of the antibacterial target PBP1b at 3.3 Å resolution.

The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of ß-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

A Slippery Scaffold: Synthesis and Recycling of the Bacterial Cell Wall Carrier Lipid.

The biosynthesis of bacterial cell envelope polysaccharides such as peptidoglycan relies on the use of a dedicated carrier lipid both for the assembly of precursors at the cytoplasmic face of the plasma membrane and for the translocation of lipid linked oligosaccharides across the plasma membrane into the periplasmic space. This dedicated carrier lipid, undecaprenyl phosphate, results from the dephosphorylation of undecaprenyl pyrophosphate, which is generated de novo in the cytoplasm by undecaprenyl pyrophosphate synthase and released as a by-product when newly synthesized glycans are incorporated into the existing cell envelope. The de novo synthesis of undecaprenyl pyrophosphate has been thoroughly characterized from a structural and mechanistic standpoint; however, its dephosphorylation to the active carrier lipid form, both in the course of de novo synthesis and recycling, has only been begun to be studied in depth in recent years. This review provides an overview of bacterial carrier lipid synthesis and presents the current state of knowledge regarding bacterial carrier lipid recycling.

IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase.

Mycobacterium tuberculosis (Mtb) grows on host-derived cholesterol during infection. IpdAB, found in all steroid-degrading bacteria and a determinant of pathogenicity, has been implicated in the hydrolysis of the last steroid ring. Phylogenetic analyses revealed that IpdAB orthologs form a clade of CoA transferases (CoTs). In a coupled assay with a thiolase, IpdAB transformed the cholesterol catabolite (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) and CoASH to 4-methyl-5-oxo-octanedioyl-CoA (MOODA-CoA) and acetyl-CoA with high specificity (kcat/Km = 5.8 ± 0.8 × 104 M-1⋅s-1). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a ß-keto-thioester, a thiolase substrate. Contrary to characterized CoTs, IpdAB exhibited no activity toward small CoA thioesters. Further, IpdAB lacks the catalytic glutamate residue that is conserved in the ß-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105A, conserved in the α-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB·COCHEA-CoA complex, solved to 1.4 Å, revealed that Glu105A is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105AA variant accumulated a yellow-colored species (λmax = 310 nm; Kd = 0.4 ± 0.2 µM) typical of ß-keto enolates. In the presence of D2O, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with kcat Based on these data and additional IpdAB variants, we propose a retro-Claisen condensation-like mechanism for the IpdAB-mediated hydrolysis of COCHEA-CoA. This study expands the range of known reactions catalyzed by the CoT superfamily and provides mechanistic insight into an important determinant of Mtb pathogenesis.

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

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.

A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA-competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro-domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C-terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C-terminal helix are required for MAF1-mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co-immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA-competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus.

Purification, crystallization and X-ray diffraction analysis of Trypanosoma congolense insect-stage surface antigen (TcCISSA).

Trypanosoma congolense is a major contributor to the vast socioeconomic devastation in sub-Saharan Africa caused by animal African trypanosomiasis. These protozoan parasites are transmitted between mammalian hosts by tsetse-fly vectors. A lack of understanding of the molecular basis of tsetse-trypanosome interactions stands as a barrier to the development of improved control strategies. Recently, a stage-specific T. congolense protein, T. congolense insect-stage surface antigen (TcCISSA), was identified that shows considerable sequence identity (>60%) to a previously identified T. brucei insect-stage surface molecule that plays a role in the maturation of infections. TcCISSA has multiple di-amino-acid and tri-amino-acid repeats in its extracellular domain, making it an especially interesting structure-function target. The predicted mature extracellular domain of TcCISSA was produced by recombinant DNA techniques, purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis; the data were processed to 2.7 Šresolution.