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The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 μg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4 μg mL-1 , the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd ≈24 μm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.

Lyme disease, the most common vector-borne illness in North America, is caused by the spirochete Borrelia burgdorferi. Infection begins in the skin following a tick bite and can spread to the hearts, joints, nervous system, and other organs. Diverse host responses influence the level of B. burgdorferi infection in mice and humans. Using a systems biology approach, we examined potential molecular interactions between human extracellular and secreted proteins and B. burgdorferi. A yeast display library expressing 1031 human extracellular proteins was probed against 36 isolates of B. burgdorferi sensu lato. We found that human Peptidoglycan Recognition Protein 1 (PGLYRP1) interacted with the vast majority of B. burgdorferi isolates. In subsequent experiments, we demonstrated that recombinant PGLYRP1 interacts with purified B. burgdorferi peptidoglycan and exhibits borreliacidal activity, suggesting that vertebrate hosts may use PGLYRP1 to identify B. burgdorferi. We examined B. burgdorferi infection in mice lacking PGLYRP1 and observed an increased spirochete burden in the heart and joints, along with splenomegaly. Mice lacking PGLYRP1 also showed signs of immune dysregulation, including lower serum IgG levels and higher levels of IFNγ, CXCL9, and CXCL10.Taken together, our findings suggest that PGLYRP1 plays a role in the host’s response to B. burgdorferi and further demonstrate the utility of expansive yeast display screening in capturing biologically relevant interactions between spirochetes and their hosts., Author summary Lyme disease is the most common vector-borne illness in North America and is caused by the spirochete Borrelia burgdorferi. The disease starts with a tick bite that leads to a skin rash and inflammation in other organs of the body, such as hearts and joints. B. burgdorferi uses many strategies to evade detection and persist in the human host. It is important to have efficient methods to be able to identify the various components of the immune system that interact with B. burgdorferi to better understand the disease process, but few currently exist. In this study, we used a novel yeast display screening assay of over 1,000 human immune proteins probed against several isolates of Borrelia to uncover biologically relevant interactions for the Lyme disease pathogen. We identified Peptidoglycan Recognition Protein 1 (PGLYRP1), an innate immune protein important in defense against bacteria, as a major candidate from this screen. We validated the interaction of PGLYRP1 with Borrelia and were able to use PGLYRP1-deficient mice as a model to understand the role of this protein in Lyme disease pathogenesis. Our study demonstrates the potential implications of yeast screens in uncovering important host-pathogen interactions.

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen.IMPORTANCEGene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

Significance Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America. If early infection is untreated, it can result in late-stage manifestations, including arthritis. Although antibiotics are generally effective at all stages of the disease, arthritis may persist in some patients for months to several years despite oral and intravenous antibiotic treatment. Excessive, dysregulated host immune responses are thought to play an important role in this outcome, but the underlying mechanisms are not completely understood. This study identifies the B. burgdorferi peptidoglycan, a major component of the cell wall, as an immunogen likely to contribute to inflammation during infection and in cases of postinfectious Lyme arthritis., Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb. In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis , which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of “database filtering” bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 μM) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria ( Streptococcus ) and Gram-negative bacteria ( Escherichia coli ). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created.

Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium’s transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents., Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging in B. burgdorferi. We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete. IMPORTANCE Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium’s transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirocheteBorrelia burgdorferi, is thus evermore pressing. Despite important advances inB. burgdorferigenetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multi-color imaging inB. burgdorferi. We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Lastly, we describe a set of constitutively active promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities inB. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, aB. burgdorferilipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites ofB. burgdorfericells, highlighting the complex subcellular organization of this spirochete.IMPORTANCEGenetic manipulation of the Lyme disease spirocheteB. burgdorferiremains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest,B. burgdorferidisplays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium’s transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use inB. burgdorferiby generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and constitutively active promoters of different strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)–based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction–fluorescent microsphere assay (LDR-FMA), we developed a multiplex assay that has the capability to simultaneously detect all four Plasmodium spp. and W. bancrofti infections in blood samples. Compared with microfilarial positivity in the blood, the LDR-FMA assay is highly concordant (91%), sensitive (86%), and specific (94%), and has high reproducibility for Plasmodium spp. (85–93%) and W. bancrofti (90%) diagnoses. The development of this assay for the simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of human blood and mosquito samples from co-endemic areas.