Genetic Manipulation of <i>Neisseria gonorrhoeae</i>

Dillard, Joseph P.

doi:10.1002/9780471729259.mc04a02s00

Cited by 75 publications

(60 citation statements)

References 47 publications

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“…To confirm that NMB0825 was indeed involved in the induction of the HIV-1 LTR, the mutation was complemented with a functional copy of the ORF. A single copy of NMB0825 was reintroduced in trans within the mutant genome under lac promoter control using the pGCC4 vector system (30). In the absence of isopropyl β-D-1-thiogalactopyranoside (IPTG), the complemented strain induced HIV-1 LTR expression at levels an average of 5.7-fold above background ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives HIV-1 expression

Malott¹,

Keller

Gaudet³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Clinical and epidemiological synergy exists between the globally important sexually transmitted infections, gonorrhea and HIV. Neisseria gonorrhoeae , which causes gonorrhea, is particularly adept at driving HIV-1 expression, but the molecular determinants of this relationship remain undefined. N. gonorrhoeae liberates a soluble factor that potently induces expression from the HIV-1 LTR in coinfected cluster of differentiation 4-positive (CD4 + ) T lymphocytes, but this factor is not a previously described innate effector. A genome-wide mutagenesis approach was undertaken to reveal which component(s) of N. gonorrhoeae induce HIV-1 expression in CD4 + T lymphocytes. A mutation in the ADP-heptose biosynthesis gene, hldA , rendered the bacteria unable to induce HIV-1 expression. The hldA mutant has a truncated lipooligosaccharide structure, contains lipid A in its outer membrane, and remains bioactive in a TLR4 reporter-based assay but did not induce HIV-1 expression. Mass spectrometry analysis of extensively fractionated N. gonorrhoeae- derived supernatants revealed that the LTR-inducing fraction contained a compound having a mass consistent with heptose-monophosphate (HMP). Heptose is a carbohydrate common in microbes but is absent from the mammalian glycome. Although ADP-heptose biosynthesis is common among Gram-negative bacteria, and heptose is a core component of most lipopolysaccharides, N. gonorrhoeae is peculiar in that it effectively liberates HMP during growth. This N. gonorrhoeae- derived HMP activates CD4 + T cells to invoke an NF-κB–dependent transcriptional response that drives HIV-1 expression and viral production. Our study thereby shows that heptose is a microbial-specific product that is sensed as an innate immune agonist and unveils the molecular link between N. gonorrhoeae and HIV-1.

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Section: Resultsmentioning

confidence: 99%

Neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives HIV-1 expression

Malott¹,

Keller

Gaudet³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Graver-Wade (GW) defined medium (pH 6.8) was used for the growth of gonococci in liquid culture for DNA secretion assays and growth rate determinations (53). For microarray experiments, gonococci were grown in GCB liquid medium with supplements as described previously (10). For plasmid selection in E. coli, chloramphenicol was used at a concentration of 25 g ml Ϫ1 and erythromycin was used at a concentration of 500 g ml…”

Section: Methodsmentioning

confidence: 99%

“…The N. gonorrhoeae strain MS11 was used as the wild-type (WT) strain and for the construction of the gonococcal strains described in Table 1. Growth of gonococci on GC medium base (GCB) agar (Difco) was performed as previously described (10). Escherichia coli strains were grown on Luria-Bertani (LB) agar plates or in LB broth (40).…”

Section: Methodsmentioning

confidence: 99%

“…The traD deletion removes bases 4 to 1,978, 97% of the coding sequence, but it maintains the predicted ribosome-binding site and start codon for the downstream gene yaa and is thus predicted not to have polar effects on yaa. The traD deletion or the traI frameshift mutation was introduced into gonococci by allelic exchange without selection (10). Complementation was achieved by introduction of the wildtype copy of traD or traI into the gonococcal chromosome between aspC and lctP along with the cat marker via the pKH37 vector (29).…”

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confidence: 99%

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Increased Expression of the Type IV Secretion System in Piliated Neisseria gonorrhoeae Variants

Salgado-Pabón

Hackett

et al. 2010

J Bacteriol

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Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.

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“…The length of the pilT flanking sequences in the amplicon was extended by PCR using primers NP252F and NP252R. The amplification products were purified and used in spot or liquid transformations (Dillard, 2006) of AP2031…”

Section: à5mentioning

confidence: 99%

Isolation and characterization of Neisseria musculi sp. nov., from the wild house mouse

Weyand

Phifer‐Rixey

et al. 2016

International Journal of Systematic and Evolutionary Microbiology

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Genetic Manipulation of Neisseria gonorrhoeae

Cited by 75 publications

References 47 publications

Neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives HIV-1 expression

Neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives HIV-1 expression

Increased Expression of the Type IV Secretion System in Piliated Neisseria gonorrhoeae Variants

Isolation and characterization of Neisseria musculi sp. nov., from the wild house mouse

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