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

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

Intravenous versus Partial Oral Antibiotic Therapy in the Treatment of Uncomplicated Bloodstream Infection Due to <i>Streptococcus</i> Species

This retrospective cohort study examines effectiveness of partial oral antibiotic regimens in uncomplicated bloodstream infections (BSIs) due to Streptococcus species compared to standard intravenous therapy. Adult patients with uncomplicated streptococcal BSIs from April 2016 to June 2020 in seven hospitals in South Carolina, USA, were evaluated. Multivariate Cox proportional hazards regression was used to examine the time to treatment failure within 90 days of a BSI after adjustment for the propensity to receive partial oral therapy. Multivariate linear regression was used to examine the hospital length of stay (HLOS). Among the 222 patients included, 99 received standard intravenous antibiotics and 123 received partial oral therapy. Of the standard intravenous therapy group, 46/99 (46.5%) required outpatient parenteral antibiotic therapy (OPAT). There was no difference in the risk of treatment failure between partial oral and standard intravenous therapy (hazards ratio 0.53, 95% CI 0.18, 1.60; p = 0.25). Partial oral therapy was independently associated with a shorter HLOS after adjustments for the propensity to receive partial oral therapy and other potential confounders (−2.23 days, 95% CI −3.53, −0.94; p < 0.001). Transitioning patients to oral antibiotics may be a reasonable strategy in the management of uncomplicated streptococcal BSIs. Partial oral therapy does not seem to have a higher risk of treatment failure and may spare patients from prolonged hospitalizations and OPAT complications

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 <i>Borrelia burgdorferi</i> genes important for resistance against reactive oxygen and nitrogen species

<div><p><i>Borrelia burgdorferi</i>, 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. <i>B</i>. <i>burgdorferi</i> contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of <i>B</i>. <i>burgdorferi</i> 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 <i>in vitro</i>. 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::<i>bb0164</i> mutant, identifying a novel role for BB0164 in <i>B</i>. <i>burgdorferi</i> manganese homeostasis. Infection of C57BL/6 and <i>gp91</i>^{<i>phox</i>-/-} mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the <i>in vitro</i> screens are required for infectivity in mice. Collectively, our data provide insight into how <i>B</i>. <i>burgdorferi</i> responds to ROS and RNS and suggests that this response is relevant to the <i>in vivo</i> success of the organism.</p></div

Obstetric and Newborn Weak D-Phenotype RBC Testing and Rh Immune Globulin Management Recommendations: Lessons From a Blinded Specimen-Testing Survey of 81 Transfusion Services

CONTEXT.—: Modern RHD genotyping can be used to determine when patients with serologic weak D phenotypes have RHD gene variants at risk for anti-D alloimmunization. However, serologic testing, RhD interpretations, and laboratory management of these patients are quite variable. OBJECTIVE.—: To obtain interlaboratory comparisons of serologic testing, RhD interpretations, Rh immune globulin (RhIG) management, fetomaternal hemorrhage testing, and RHD genotyping for weak D-reactive specimens. DESIGN.—: We devised an educational exercise in which 81 transfusion services supporting obstetrics performed tube-method RhD typing on 2 unknown red blood cell challenge specimens identified as (1) maternal and (2) newborn. Both specimens were from the same weak D-reactive donor. The exercise revealed how participants responded to these different clinical situations. RESULTS.—: Of reporting laboratories, 14% (11 of 80) obtained discrepant immediate-spin reactions on the 2 specimens. Nine different reporting terms were used to interpret weak D-reactive maternal RhD types to obstetricians. In laboratories obtaining negative maternal immediate-spin reactions, 28% (16 of 57) performed unwarranted antiglobulin testing, sometimes leading to recommendations against giving RhIG. To screen for excess fetomaternal hemorrhage after a weak D-reactive newborn, 47% (34 of 73) of reporting laboratories would have employed a contraindicated fetal rosette test, risking false-negative results and inadequate RhIG coverage. Sixty percent (44 of 73) of laboratories would obtain RHD genotyping in some or all cases. CONCLUSIONS.—: For obstetric and neonatal patients with serologic weak D phenotypes, we found several critical problems in transfusion service laboratory practices. We provide recommendations for appropriate testing, consistent immunohematologic terminology, and RHD genotype-guided management of Rh immune globulin therapy and RBC transfusions

Confirmation of ROS sensitivity.

<p>Individual Tn mutants, complemented mutants, and the parental strain were exposed to H₂O₂, TBHP, or culture medium alone. Bacterial cell density was quantified by dark-field microscopy following a three-day outgrowth period. An outgrowth ratio was determined for each strain as the ratio of cell numbers in the treated sample compared to the untreated sample. Results for 125 μM H₂O₂ (A) or 7.5 mM TBHP (B) are shown. *, P < 0.01 compared to 5A18NP1; §, P < 0.05 compared to 5A18NP1 by one-way ANOVA followed by Dunnett’s test. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.s009" target="_blank">S5 Table</a> for Tn clone numbers and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.s010" target="_blank">S6 Table</a> for a description of the complemented Tn mutants (Tn::<i>bb0017</i>-comp, DM104; Tn::<i>bb0164</i>-comp, JH511).</p

Putative <i>B</i>. <i>burgdorferi</i> genes involved in RNS and ROS resistance.

<p>Putative <i>B</i>. <i>burgdorferi</i> genes involved in RNS and ROS resistance.</p