A fully defined, clear and protein-free liquid medium permitting dense growth of<i>Neisseria gonorrhoeae</i>from very low inocula

Wade, J. J.; Graver, Michelle Angela

doi:10.1111/j.1574-6968.2007.00776.x

Cited by 55 publications

(48 citation statements)

References 12 publications

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“…Piliated colonies were used for transformation experiments, and nonpiliated colonies were used in all other experiments. To initiate growth in liquid medium, nonpiliated colonies were collected from plates incubated for ϳ18 h and suspended to an OD 600 of 0.1 in prewarmed GCB liquid (GCBL) medium supplemented as described above with the addition of 0.042% sodium bicarbonate, or in Graver-Wade (GW) medium (51). Suspensions in GCBL medium were incubated at 37°C with shaking at 220 rpm for 3 h. GW medium suspensions were cultured in the same manner in a 5% CO 2 environment.…”

Section: Methodsmentioning

confidence: 99%

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

et al. 2017

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The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomicsdriven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344 -in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.KEYWORDS Neisseria gonorrhoeae, vaccine antigens, cell envelope, phenotypic microarrays, antibiotics, Biolog I n Gram-negative bacteria, the cell envelope is composed of the outer membrane, the cell wall, and the cytoplasmic or inner membrane, each of which can be further broken down into its constituents (1). The outer membrane is a bilayer composed of a lipopolysaccharide (LPS) or lipooligosaccharide (LOS) outer leaflet facing the extracel-

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Section: Methodsmentioning

confidence: 99%

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

et al. 2017

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“…The SJ-GC medium contained potato starch, a known drug binder, so the chemically defined Graver-Wade (GW) medium was used for MIC determination for both N. gonorrhoeae and N. meningitidis. The GW medium was made as described (34). Bovine serum albumin (10 mg/ml) was added to the GW medium to increase the fatty acid binding capacity of the medium.…”

Section: Methodsmentioning

confidence: 99%

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

Yao

Bruhn

Frank

et al. 2016

Journal of Biological Chemistry

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Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.Neisseria is a genus of mucosal colonizing bacteria found in a variety of animals. Of the 11 species of Neisseria associated with humans, Neisseria gonorrhoeae and Neisseria meningitidis are obligate human pathogens. N. meningitidis is a common cause of meningitis and other meningococcal diseases in infants and children with 3,000 cases/year in the United States (1). N. gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhoeae, with Ͼ100 million new cases/year worldwide (2). The incidence of multidrug-resistant N. gonorrhoeae has markedly increased in recent years, raising the prospect of untreatable gonorrhoeae and highlighting the need for developing new antibiotics and discovering new antibiotic targets in Neisseria (2). One emerging antibiotic drug target is bacterial type II fatty acid synthesis (FASII) 3 (3-6). All characterized bacteria have the ability to utilize exogenous fatty acids for phospholipid synthesis (7); thus, it is important to understand the pathways for fatty acid uptake and whether extracellular fatty acids can bypass FASII inhibition (7-9).Neisseria are Gram-negative bacteria with phospholipids containing saturated and unsaturated fatty acids (10) and LPS containing 3-hydroxy fatty acids (11). Escherichia coli utilizes an acyl-CoA synthetase (FadD) to activate exogenous fatty acids (12), and they are incorporated into phospholipids by the PlsB/PlsC acyltransferase system that utilizes either acyl-ACP or acyl-CoA (7, 13). E. coli cannot bypass FASII inhibitors because there is no mechanism for the generation of acyl-ACP from extracellular fatty acids, and FASII is the only source for the 3-hydroxy fatty acids required to synthesize the essential LPS (14, 15). The fatty acid composition of Neisseria resembl...

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“…Routine culture of N. gonorrhoeae was conducted using gonococcal medium base (GCB) (Difco) at 37°C in 5% CO 2 or in GCB liquid broth containing Kellogg supplements (24). GW is a fully defined liquid medium (25) that was used to test the response of N. gonorrhoeae strains to SP and other compounds, when indicated. Pooled human semen was purchased from both Lee Biosolutions and Innovative Research.…”

Section: Methodsmentioning

confidence: 99%

Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation

et al. 2016

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Neisseria gonorrhoeae causes the human-specific disease gonorrhea and is transmitted from person to person primarily via sexual contact. During transmission, N. gonorrhoeae is often exposed to seminal fluid and must adapt to this change in environment. Previous work demonstrated that seminal fluid facilitates N. gonorrhoeae motility and alters epithelial cell interactions. In this study, exposure to seminal fluid was found to decrease surface adherence of gonococci in a manner that was independent of Opa adhesin proteins or type IV pilus retraction. Semen was also shown to cause dispersal of bacteria that had previously established surface adherence. Although surface adherence decreased, interbacterial interactions were increased by seminal plasma both in long-term static culture and on a cell-to-cell basis over shorter time periods. The result of increased bacterium-bacterium interactions resulted in the formation of microcolonies, an important step in the N. gonorrhoeae infectious process. Seminal fluid also facilitated increased bacterial aggregation in the form of shear-resistant three-dimensional biofilms. These results emphasize the importance of the gonococcal response to the influx of seminal fluid within the genital niche. Further characterization of the N. gonorrhoeae response to semen will advance our understanding of the mechanisms behind the establishment of infection in naive hosts and the process of transmission. IMPORTANCE N. gonorrhoeae is the causative agent of the globally prevalent sexually transmitted infection gonorrhea. An understudied aspect of this human-adapted pathogen is the change in bacterial physiology that occurs during sexual transmission. N. gonorrhoeae encounters semen when transmitted from host to host, and it is known that, when N. gonorrhoeae is exposed to seminal fluid, alterations in bacterial motility and type IV pilus arrangement occur. This work extends our previous observations on this modulation of gonococcal physiology by seminal fluid and demonstrates that seminal plasma decreases surface adherence, promotes interbacterial interactions, and enhances biofilm formation.

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A fully defined, clear and protein-free liquid medium permitting dense growth ofNeisseria gonorrhoeaefrom very low inocula

Cited by 55 publications

References 12 publications

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation

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