Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.

A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.

TraK and TraB are conserved outer membrane proteins of the Neisseria gonorrhoeae Type IV secretion system and are expressed at low levels in wild-type cells.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.

Characterization of SARS-CoV-2 Aerosols Dispersed During Noninvasive Respiratory Support of Patients With COVID-19.

BACKGROUND: In the midst of the COVID-19 pandemic, noninvasive respiratory support (NRS) therapies such as high-flow nasal cannula (HFNC) and noninvasive ventilation (NIV) were central to respiratory care. The extent to which these treatments increase the generation and dispersion of infectious respiratory aerosols is not fully understood. The objective of this study was to characterize SARS-CoV-2 aerosol dispersion from subjects with COVID-19 undergoing NRS therapy. METHODS: Several different aerosol sampling devices were used to collect air samples in the vicinity of 31 subjects with COVID-19, most of whom were receiving NRS therapy, primarily HFNC. Aerosols were collected onto filters and analyzed for the presence of SARS-CoV-2 RNA. Additional measurements were collected in an aerosol chamber with healthy adult subjects using respiratory therapy devices under controlled and reproducible conditions. RESULTS: Fifty aerosol samples were collected from subjects receiving HFNC or NIV therapy, whereas 6 samples were collected from subjects not receiving NRS. Only 4 of the 56 aerosol samples were positive for SARS-CoV-2 RNA, and all positive samples were collected using a high air flow scavenger mask collection device placed in close proximity to the subject. The chamber measurements with healthy subjects did not show any significant increase in aerosol dispersion caused by the respiratory therapy devices compared to baseline. CONCLUSIONS: Our findings demonstrate very limited detection of SARS-CoV-2-containing aerosols in the vicinity of subjects with COVID-19 receiving NRS therapies in the clinical setting. These results, combined with controlled chamber measurements showing that HFNC and NIV device usage was not associated with increased aerosol dispersion, suggest that NRS therapies do not result in increased dispersal of aerosols in the clinical setting.

New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis.

We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-ß-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.

Control of the type 3 secretion system in Vibrio harveyi by quorum sensing through repression of ExsA.

The type 3 secretion system (T3SS) genes of Vibrio harveyi are activated at low cell density and repressed at high cell density by quorum sensing (QS). Repression requires LuxR, the master transcriptional regulator of QS-controlled genes. Here, we determine the mechanism underlying the LuxR repression of the T3SS system. Using a fluorescence-based cell sorting approach, we isolated V. harveyi mutants that are unable to express T3SS genes at low cell density and identified two mutations in the V. harveyi exsBA operon. While LuxR directly represses the expression of exsBA, complementation and epistasis analyses reveal that it is the repression of exsA expression, but not exsB expression, that is responsible for the QS-mediated repression of T3SS genes at high cell density. The present work further defines the genes in the V. harveyi QS regulon and elucidates a mechanism demonstrating how multiple regulators can be linked in series to direct the expression of QS target genes specifically at low or high cell density.

Minimal genomes, maximal productivity: comparative genomics of the photosystem and light-harvesting complexes in the marine cyanobacterium, Prochlorococcus.

Although Prochlorococcus isolates possess the smallest genomes of any extant photosynthetic organism, this genus numerically dominates vast regions of the world's subtropical and tropical open oceans and has evolved to become an important contributor to global biogeochemical cycles. The sequencing of 12 Prochlorococcus genomes provides a glimpse of the extensive genetic heterogeneity and, thus, physiological potential of the lineage. In this study, we present an up-to-date comparative analysis of major proteins of the photosynthetic apparatus in 12 Prochlorococcus genomes. Our analyses reveal a striking diversity within the Prochlorococcus lineage in the major protein complexes of the photosynthetic apparatus. The heterogeneity that has evolved in the photosynthetic apparatus suggests versatility in strategies for optimizing photosynthesis under conditions of environmental variability and stress. This diversity could be particularly important in ensuring the survival of a lineage whose individuals have evolved minimal genomes and, thus, relatively limited repertoires for responding to environmental challenges.

The Gonococcal Genetic Island and Type IV Secretion in the Pathogenic Neisseria.

Eighty percent of Neisseria gonorrhoeae strains and some Neisseria meningitidis strains encode a 57-kb gonococcal genetic island (GGI). The GGI was horizontally acquired and is inserted in the chromosome at the replication terminus. The GGI is flanked by direct repeats, and site-specific recombination at these sites results in excision of the GGI and may be responsible for its original acquisition. Although the role of the GGI in N. meningitidis is unclear, the GGI in N. gonorrhoeae encodes a type IV secretion system (T4SS). T4SS are versatile multi-protein complexes and include both conjugation systems as well as effector systems that translocate either proteins or DNA-protein complexes. In N. gonorrhoeae, the T4SS secretes single-stranded chromosomal DNA into the extracellular milieu in a contact-independent manner. Importantly, the DNA secreted through the T4SS is effective in natural transformation and therefore contributes to the spread of genetic information through Neisseria populations. Mutagenesis experiments have identified genes for DNA secretion including those encoding putative structural components of the apparatus, peptidoglycanases which may act in assembly, and relaxosome components for processing the DNA and delivering it to the apparatus. The T4SS may also play a role in infection by N. gonorrhoeae. During intracellular infection, N. gonorrhoeae requires the Ton complex for iron acquisition and survival. However, N. gonorrhoeae strains that do not express the Ton complex can survive intracellularly if they express structural components of the T4SS. These data provide evidence that the T4SS is expressed during intracellular infection and suggest that the T4SS may provide an advantage for intracellular survival. Here we review our current understanding of how the GGI and type IV secretion affect natural transformation and pathogenesis in N. gonorrhoeae and N. meningitidis.