Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of the sexually transmitted infection gonorrhea. The pathogen is a global health challenge since no protective immunity results from infection, and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising vaccine antigens as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs is part of the hemoglobin transport system comprised of HpuA and HpuB. This system allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHb, although the growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme-binding motifs and a hypothetical α-helical region, both of which may be important for the use of hHb. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb and extracting and internalizing iron.

Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization.

Neisseria gonorrhoeae is the etiological agent of the sexually-transmitted infection gonorrhea and a global health challenge since no protective immunity results from infection and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising antigen targets as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs, the hemoglobin transport system comprised of HpuA and HpuB, allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHB, although their growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme binding motifs and a hypothetical α-helical region. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb, and extracting and internalizing iron.

Acclimation to Nutritional Immunity and Metal Intoxication Requires Zinc, Manganese, and Copper Homeostasis in the Pathogenic Neisseriae.

Neisseria gonorrhoeae and Neisseria meningitidis are human-specific pathogens in the Neisseriaceae family that can cause devastating diseases. Although both species inhabit mucosal surfaces, they cause dramatically different diseases. Despite this, they have evolved similar mechanisms to survive and thrive in a metal-restricted host. The human host restricts, or overloads, the bacterial metal nutrient supply within host cell niches to limit pathogenesis and disease progression. Thus, the pathogenic Neisseria require appropriate metal homeostasis mechanisms to acclimate to such a hostile and ever-changing host environment. This review discusses the mechanisms by which the host allocates and alters zinc, manganese, and copper levels and the ability of the pathogenic Neisseria to sense and respond to such alterations. This review will also discuss integrated metal homeostasis in N. gonorrhoeae and the significance of investigating metal interplay.

Mutagenesis of the Loop 3 α-Helix of Neisseria gonorrhoeae TdfJ Inhibits S100A7 Binding and Utilization.

Neisseria gonorrhoeae causes the sexually transmitted infection (STI) gonorrhea, which afflicts over 80 million people each year. No vaccine is available to prevent gonorrhea. The pathogen alters the expression and antigenic presentation of key surface molecules, making the identification of suitable vaccine targets difficult. The human host utilizes metal-binding proteins to limit free essential transition metal ions available to invading pathogens, limiting their infective potential, a process called nutritional immunity. To overcome this, N. gonorrhoeae employs outer membrane TonB-dependent transporters (TdTs) that bind host nutritional immunity proteins and strip them of their metal cargo. The TdTs are well conserved, and some play key roles in establishing infections, making them promising vaccine targets. One TdT, TdfJ, recognizes human S100A7, a zinc-binding protein that inhibits the proliferation of other pathogens via zinc sequestration. N. gonorrhoeae uses TdfJ to strip and internalize zinc from S100A7. TdfJ contains a conserved α-helix finger in extracellular loop 3; a similar α-helix in loop 3 of another gonococcal TdT, TbpA, plays a critical role in the interaction between TbpA and human transferrin. Therefore, we hypothesized that the TdfJ loop 3 helix (L3H) participates in interactions with S100A7. We determined the affinity between wild-type TdfJ and S100A7 and then generated a series of mutations in the TdfJ L3H. Our study revealed that mutagenesis of key residues within the L3H reduced S100A7 binding and zinc piracy by the gonococcus, with profound effects seen with substitutions at residues K261 and R262. Taken together, these data suggest a key role for the TdfJ L3H in subverting host metal restriction. IMPORTANCE Gonorrhea is a global threat to public health due to the increasing incidence of antimicrobial drug resistance, rising treatment costs, and lack of a protective vaccine. The prospect of untreatable gonococcal infections has spurred efforts to identify targets for novel therapeutic and prevention strategies, and members of the family of outer membrane TonB-dependent metal transporters have emerged as promising candidates. These conserved surface molecules play a critical role in establishing infection by facilitating nutrient uptake in the human host that dedicates considerable efforts to restricting nutrient availability. In this study, we characterized the binding interaction between the zinc importer TdfJ and its human zinc source, S100A7. We went on to identify a key region of TdfJ that mediates this interaction. With a more thorough understanding of the intricate relationships between these bacterial nutrient receptors and their host nutrient sources, we may help pave the way toward identifying effective prophylaxis and treatment for an important human disease.

Mutagenesis of Vibrio fischeri and Other Marine Bacteria Using Hyperactive Mini-Tn5 Derivatives.

Mutagenizing bacterial genomes with selectable transposon insertions is an effective approach for identifying the genes underlying important phenotypes. Specific bacteria may require different tools and methods for effective transposon mutagenesis, and here we describe methods to mutagenize Vibrio fischeri using an engineered mini-Tn5 transposon with synthetic "mosaic" transposon ends. The transposon is delivered by conjugation on a plasmid that cannot replicate in V. fischeri and that encodes a hyperactive transposase outside the transposon itself. The chromosomal location of insertions can be readily identified by cloning and/or PCR-based methods described here. Although developed in V. fischeri, these tools and methods have proven effective in some other bacteria as well.

An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter.

Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal "on" expression when PhoB is active, minimal background in the "off" state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at 19 additional intervening nucleotide positions for which we did not predict sequence-specific effects, the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri, enabling us to identify bases at particular randomized positions that significantly correlated with high-level "on" or low-level "off" expression. A second round of promoter design rationally constrained 13 additional positions, leading to a reporter with high-level PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized variants of green fluorescent protein (GFP), the latter of which has a half-life of 81 min in V. fischeri In culture, PhoB induced the reporter when phosphate was depleted to a concentration below 10 µM. During symbiotic colonization of its host squid, Euprymna scolopes, the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other members of the Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and reengineering.IMPORTANCE Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between the "on" and "off" states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over 34 billion sequence variants, we identified bases correlated with favorable performance by screening only 83 candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri This promoter design strategy will facilitate the engineering of other regulator-specific reporters.

Malonate degradation in Acinetobacter baylyi ADP1: operon organization and regulation by MdcR.

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of mdc genes that enable a soil bacterium, Acinetobacter baylyi ADP1, to use malonate as a carbon source. Despite previous assumptions that the mdc-gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of mdcR, a regulatory gene that is convergent to other mdc genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate mdc-gene transcription. Although mdcR deletion prevented growth on malonate, a 1 nt substitution in the promoter of mdcA enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5'-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in A. baylyi ADP1.

An Expanded Transposon Mutant Library Reveals that Vibrio fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna scolopes.

Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg2+ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.

Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri.

Proteobacteria often co-ordinate responses to carbon sources using CRP and the second messenger cyclic 3', 5'-AMP (cAMP), which combine to control transcription of genes during growth on non-glucose substrates as part of the catabolite-repression response. Here we show that cAMP-CRP is active and important in Vibrio fischeri during colonization of its host squid Euprymna scolopes. Moreover, consistent with a classical role in catabolite repression, a cAMP-CRP-dependent reporter showed lower activity in cells grown in media amended with glucose rather than glycerol. Surprisingly though, intracellular cAMP levels were higher in glucose-grown cells. Mutant analyses were consistent with predictions that CyaA was responsible for cAMP generation, that the EIIA(Glc) component of glucose transport could enhance cAMP production and that the phophodiesterases CpdA and CpdP consumed intracellular and extracellular cAMP respectively. However, the observation of lower cAMP levels in glycerol-grown cells seemed best explained by changes in cAMP export, via an unknown mechanism. Our data also indicated that cAMP-CRP activity decreased during growth on glucose independently of crp's native transcriptional regulation or cAMP levels. We speculate that some unknown mechanism, perhaps carbon-source-dependent post-translational modulation of CRP, may help control cAMP-CRP activity in V.fischeri.