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The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin's multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.

The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching) and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate -induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.

The cellular pathways that Epstein-Barr virus (EBV) manipulates in order to effect its lifelong persistence within hosts and facilitate its transmission between hosts are not well understood. The EBV nuclear antigen 3 (EBNA-3) family of latent infection proteins consists of transcriptional regulators that influence viral and cellular gene expression in EBV-infected cells. To identify EBNA-3B- and EBNA-3C-regulated cellular genes potentially important for virus infection in vivo, we studied a lymphoblastoid cell line (LCL) infected with an unusual EBV mutant, where a genetic manipulation to delete EBNA-3B also resulted in a significant decrease in EBNA-3C expression and slower than normal growth (3B−/3Clow). Transcriptional profiling was performed on the 3B−/3ClowLCLs, and comparison of mutant and wild-type LCL profiles resulted in a group of 21 probe sets representing 16 individual genes showing statistically significant differences in expression. Further quantitative reverse transcription-PCR analyses comparing 3B−/3ClowLCLs to a previously described EBNA-3B mutant (3B−) where EBNA-3C expression was normal revealed three potential EBNA-3B-repressed genes, three potential EBNA-3C-repressed genes, and two potential EBNA-3C-activated genes. The most highly EBNA-3C-repressed gene was Jagged1, a cell surface ligand and inducer of the Notch receptor signaling pathway that is usurped by EBV genes essential for B-cell immortalization. 3B−/3ClowLCLs expressed increased levels of Jagged1 protein and were able to more efficiently induce functional Notch signaling, and this signaling was dependent on Notch cleavage by γ-secretase. However, inhibiting γ-secretase-mediated Notch cleavage did not rescue 3B−/3ClowLCL growth, suggesting that EBNA-3C-mediated repression of this signaling pathway did not contribute to LCL growth in tissue culture. Similarly, expression of the chemokine receptor CXCR4 was reproducibly upregulated in EBNA-3B-null LCLs. Since deletion of EBNA-3B has no significant impact on B-cell immortalization in tissue culture, this finding suggested that EBNA-3B-mediated regulation of CXCR4 may be an important viral strategy for alteration of B-cell homing in the infected host. These studies identify two cellular genes that do not contribute to EBV-induced B-cell growth but whose expression levels are strongly EBNA-3 regulated in EBV-infected primary B cells. These EBV-manipulated cellular pathways may be important for virus survival or transmission in humans, and their independence from EBV-induced B-cell growth makes them potential targets for testing in vivo with the rhesus lymphocryptovirus animal model for EBV infection.

The Epstein-Barr virus (EBV) nuclear antigen 3B (EBNA-3B) is considered nonessential for EBV-mediated B-cell growth transformation in vitro based on three virus isolates with EBNA-3B mutations. Two of these isolates could potentially express truncated EBNA-3B products, and, similarly, we now show that the third isolate, IB4, has a point mutation and in-frame deletion of 263 amino acids. In order to test whether a virus with EBNA-3B completely deleted can immortalize B-cell growth, we first cloned the EBV genome as a bacterial artificial chromosome (BAC) and showed that the BAC-derived virus was B-cell immortalization competent. Deletion of the entire EBNA-3B open reading frame from the EBV BAC had no adverse impact on growth of EBV-immortalized B cells, providing formal proof that EBNA-3B is not essential for EBV-mediated B-cell growth transformation in vitro.

Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression.

The human pathogen Neisseria gonorrhoeae recruits and interacts extensively with polymorphonuclear leukocytes (PMNs) during infection. N. gonorrhoeae is able to survive the bactericidal activity of these innate immune cells and can actively modulate PMN functions in vitro . PMNs are short-lived cells which readily undergo apoptosis, and thus the effect of N. gonorrhoeae infection on PMN survival has implications for whether PMNs might serve as an important site of bacterial replication during infection. We developed and validated an HL-60 myeloid leukemia cell culture model for PMN infection and used both these cells and primary PMNs to show that N. gonorrhoeae infection alone does not induce apoptosis and furthermore that N. gonorrhoeae can inhibit both spontaneous apoptosis and apoptosis induced by the intrinsic and extrinsic apoptosis inducers staurosporine (STS) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), respectively. N. gonorrhoeae infection also results in the activation of NF-κB signaling in neutrophils and induces secretion of an identical profile of proinflammatory cytokines and chemokines in both HL-60 cells and primary PMNs. Our data show that the HL-60 cell line can be used to effectively model N. gonorrhoeae- PMN interactions and that N. gonorrhoeae actively inhibits apoptosis induced by multiple stimuli to prolong PMN survival and potentially facilitate bacterial survival, replication, and transmission.

Neisseria meningitidis is an obligate human commensal organism that usually resides within the nasopharynx. The first step in establishing colonization of the nasopharyngeal mucosal surface is adherence to epithelial cells via a number of bacterial surface structures. Type IV pili play an important role in mediating bacterial adherence to host cells, as isolates from both asymptomatic carriers and patients with meningococcal disease are found to be heavily piliated, and piliated bacteria attach more efficiently to epithelial cells than non-piliated isolates. Subsequent to attachment, bacteria multiply on the cell surface in tightly associated microcolonies, which are highly dynamic structures that grow, fuse, and are motile (Higashi et al., 2007), and from where it is thought bacteria eventually detach to either disseminate to new locations within the existing host (e.g., via crossing the epithelial layer to access the bloodstream and the meninges), or transmit to new hosts. Type IV pili are long filamentous appendages containing multiple protein subunits, the major component of which is the main pilus subunit (pilin). Individual pili can aggregate to form bundles composed of 8–10 individual fibers, which act as coordinated retractable units that are capable of exerting nanonewton scale forces on the infected cell (Biais et al., 2008). The extent of pilus bundling appears to affect the degree of bacterial adherence to cells, as well as the propensity to grow in microcolonies (Marceau et al., 1995). Pilin subunits can be post-translationally modified by phosphoethanolamine (PE), phosphocholine (PC), phosphoglycerol (PG), and by O-linked glycosylation. Several serine residues within the N. meningitidis pilin, as well as pilin from the closely related N. gonorrhoeae, have been shown to undergo modification. Serine 63 is modified by O-linked glycosylation, serine 68 is modified by either PE or PC, and PG has been identified as a modification of serine 93 (Stimson et al., 1995, 1996; Forest et al., 1999; Hegge et al., 2004). These post-translational modifications in turn exert an influence on pilus morphology and pilus-mediated adherence to human cells, as loss of glycosylation increases N. meningitidis adherence (Marceau et al., 1998), and loss of the PE/PC modification results in increased pilus bundling in N. gonorrhoeae, but does not affect adherence (Forest et al., 1999). The genes involved in mediating glycosylation and PE/PC addition have been identified (Warren and Jennings, 2003; Hegge et al., 2004), but the enzyme responsible for PG addition was unknown. Chamot-Rooke et al. (2011) identified a N. meningitidis gene which is upregulated several-fold upon host cell contact, and which is responsible for adding phosphoglycerol (PG) to pilin subunits at serine residue 93. Mutation of the pilin phosphotransferase B (pptB) gene (NMV_0885), an ortholog of other phosphoglycerol transferases, resulted in a total loss of PG modification, and induction of a pptB gene under lac promoter control resulted in increased PG modification. Molecular modeling of the region surrounding serine 93 suggests that it carries a highly positive charge, which would be disturbed by the addition of a negatively charged PG group, which in turn was predicted to destabilize interactions between adjacent pilus fibers within a bundle. Consistent with this hypothesis, the induction of PptB expression and PG modification resulted in a decrease in pilus bundling, and a transition to expression of single pili. This decrease in bundling led to decreased bacterial aggregation, which was reversed when PG modification was abrogated by either deleting pptB or mutating the modified serine residue. Alterations in the ability of N. meningitidis to form bundled pili and to adhere to host cells has implications for its ability to both grow in microcolonies on the infected cell surface, and its ability to detach from cells to mediate dissemination. PG modification did not appear to have an effect on either bacterial attachment or microcolony growth, but deletion of pptB did result in decreased bacterial detachment from epithelial cells in a laminar flow chamber, as well as decreased cellular invasion and migration across a monolayer. These results suggest a scenario where pptB upregulation upon host cell contact triggers PG modification on pilin subunits, where the introduction of a destabilizing negative charge mediates disruption of pilus bundles and facilitates bacterial detachment from the microcolony (Figure ​(Figure1).1). This intriguing finding raises a number of additional questions: (1) What is the mechanism of pilin modification? Where is PptB localized within the bacterial cell, and are the pilin subunits modified inside the cell and subsequently translocated across the pilus biogenesis machinery? Is pilus retraction involved in regulating glycerophosphorylation? (2) What are the signals regulating pptB expression? Does pptB induction result from direct cell contact or are there other signals from the host? Is there a spatial or temporal subset of bacteria in a microcolony that receive the signal from the host cell? Is pptB expression downregulated after bacterial detachment to allow the re-formation of pilus bundles? Is pptB only induced in certain host environments and/or tissues or is the transition from bundled to unbundled pili also important for crossing the blood-brain barrier? (3) PptB is part of a regulon induced upon host cell contact that includes other genes involved in adhesion such as pilC1 and crgA. How does pptB induction fit into the broader context of regulated pilus expression during infection? (4) Do the other pilin post-translational modifications contribute to this process? Both glycosylation and PE/PC modification on other residues can affect bacterial attachment and pilus bundling, therefore are these various modifications coordinately regulated during the course of infection? If so, is there an additive effect of the modifications or a hierarchy of effects? Figure 1 The role of PptB in mediating meningococcal dissemination. (A) Bacteria growing on the epithelial cell surface in microcolonies are induced to express PptB after host cell contact, which induces addition of phosphoglycerol (PG) onto pilin subunits. (B) ... A number of other bacteria also express type IV pili, including human pathogens such as Pseudomonas aeruginosa and enteropathogenic Escherichia coli. Post-translational modification of other type IV pilus subunits has not been extensively characterized, thus it is an open question whether other pathogens that use type IV pili for attachment to host cell surfaces also employ similar mechanisms to regulate aspects of their pathogenicity. Regulated post-translational modification of any surface structures involved in mediating bacterial-host interactions could therefore provide an additional means by which bacteria fine-tune their physiology to best exploit the host environment in which they reside.