fluorescence-microscopy-methods-for-determining-viability-bacteria

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

University of Virginia Health Sciences Center

Salmonella enterica serovar Typhimurium can infect epithelial cells via the basolateral surface after breaching the intestinal epithelium, yet little is known about this process. Here, we show that actin polymerization driven by the Arp2/3 complex is critical to both basolateral and apical bacterial invasion of polarized MDCK cells. While there is also a dependence upon toxin B-sensitive Rho GTPases, none of the four GTPases known to be activated by S. enterica serovar Typhimurium SopE are individually required for basolateral internalization. These results underscore that the specific factors required for Salmonella invasion differ between membrane domains of polarized epithelia.

Coordinate regulation of Salmonella enterica serovar Typhimurium invasion of epithelial cells by the Arp2/3 complex and Rho GTPases.

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non-selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac-regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4x10(-3) events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non-piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.

The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae.

Symptomatic gonococcal infection, caused by the pathogen Neisseria gonorrhoeae (Gc), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess several mechanisms of oxidative killing, intact Gc can be found associated with PMNs, suggesting that gonococcal defences against oxidative stress are crucial for its ability to evade killing by PMNs. We used microarrays to identify genes that were differentially expressed after transient exposure of Gc to hydrogen peroxide (H2O2). Of the 75 genes found to be upregulated after H2O2 treatment, over one-quarter, including two of the most highly upregulated genes (NGO1686 and NGO554), were predicted to encode proteins with unknown functions. Further characterization of a subset of these upregulated genes demonstrated that NGO1686, a putative zinc metalloprotease, protects against oxidative damage caused by both H2O2 and cumene hydroperoxide, and that NGO554, a Gc-specific protein, acts to protect against damage caused by high levels of H2O2. Our current study also ascribes a role in H2O2 damage protection to recN, a gene previously characterized for its role in DNA repair. A PMN survival assay demonstrated that the recN and NGO1686 mutants were more susceptible to killing than the parent strain FA1090. These results define for the first time the robust transcriptional response to H2O2 by this strict human pathogen and underscore the importance of this system for survival to host defences.

The transcriptome response of Neisseria gonorrhoeae to hydrogen peroxide reveals genes with previously uncharacterized roles in oxidative damage protection.

Type IV pili are a major virulence factor of the obligate human pathogen Neisseria gonorrhoeae (the gonococcus; Gc). Pili facilitate bacterial adherence to epithelial cells, but their participation in later steps of epithelial infection, particularly intracellular replication and exit, is poorly understood. Using polarized T84 cells as a model for mature mucosal epithelia, pilus dynamics in piliated, Opa-expressing Gc were examined over time. T84 infection was characterized by a several-hour delay in the growth of cell-associated bacteria and by non-directional exit of Gc, the first time these phenomena have been reported. During infection, non-piliated progeny arose stochastically from piliated progenitors. Piliated and non-piliated Gc replicated and exited from T84 cell monolayers equally well, demonstrating that piliation did not influence Gc survival during epithelial infection. The frequency with which pilin variants arose from a defined piliated progenitor during T84 cell infection was found to be sufficiently high to account for the extensive pilin variation reported during human infection. However, the repertoire of variants appearing in association with T84 cells was similar to what was seen in the absence of cells, demonstrating that polarized epithelial cells can support Gc replication without selecting for a subset of pilin variants or piliation states.

Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili.

Gene conversion mediates the variation of virulence-associated surface structures on pathogenic microorganisms, which prevents host humoral immune responses from being effective. One of the best-studied gene conversion systems is antigenic variation (Av) of the pilin subunit of the Neisseria gonorrhoeae type IV pilus. To identify cis-acting DNA sequences that facilitate Av, the 700-bp region upstream of the pilin gene pilE was targeted for transposon mutagenesis. Four classes of transposon-associated mutations were isolated, distinguishable by their pilus-associated phenotypes: (i) insertions that did not alter Av or piliation, (ii) insertions that blocked Av, (iii) insertions that interfered with Av, and (iv) insertions that interfered with pilus expression and Av. Mutagenesis of the pilE promoter did not affect the frequency of Av, directly demonstrating that pilin Av is independent of pilE transcription. Two stretches of sequence upstream of pilE were devoid of transposon insertions, and some deletions in these regions were not recoverable, suggesting that they are essential for gonococcal viability. Insertions that blocked pilin Av were located downstream of the RS1 repeat sequence, and deletion of the region surrounding these insertions completely abrogated pilin Av, confirming that specific sequences 5' to pilE are essential for the recombination events underlying pilin Av.

Transposon mutagenesis identifies sites upstream of the Neisseria gonorrhoeae pilE gene that modulate pilin antigenic variation.

Symptomatic infection with Neisseria gonorrhoeae (Gc) results in a potent polymorphonuclear leukocyte (PMN)-driven inflammatory response, but the mechanisms by which Gc withstands PMN attack are poorly defined. Here we report that Gc can suppress the PMN oxidative burst, a central component of the PMN antimicrobial arsenal. Primary human PMNs remained viable after exposure to liquid-grown, exponential-phase, opacity-associated protein (Opa)-negative Gc of strains FA1090 and MS11 but did not generate reactive oxygen species (ROS), even after bacterial opsonization. Liquid-grown FA1090 Gc expressing OpaB, an Opa protein previously correlated with PMN ROS production, elicited a minor PMN oxidative burst. PMN ROS production in response to Opa(-) and OpaB+ Gc was markedly enhanced if bacteria were agar-grown or if liquid-grown bacteria were heat-killed. Liquid-grown Opa(-) Gc inhibited the PMN oxidative burst elicited by isogenic dead bacteria, formylated peptides or Staphylococcus aureus but did not inhibit PMN ROS production by OpaB+ Gc or phorbol esters. Suppression of the oxidative burst required Gc-PMN contact and bacterial protein synthesis but not phagocytosis. These results suggest that viable Gc directly inhibits PMN signalling pathways required for induction of the oxidative burst, which may contribute to gonococcal pathogenesis during inflammatory stages of gonorrhoeal disease.

Neisseria gonorrhoeae suppresses the oxidative burst of human polymorphonuclear leukocytes.

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40-70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro, had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.

Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes.

The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen.

Mismatch correction modulates mutation frequency and pilus phase and antigenic variation in Neisseria gonorrhoeae.

Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen.

Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation.

The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation.

Infection with the human-specific bacterial pathogen Neisseria gonorrhoeae triggers a potent, local inflammatory response driven by polymorphonuclear leukocytes (neutrophils or PMNs). PMNs are terminally differentiated phagocytic cells that are a vital component of the host innate immune response and are the first responders to bacterial and fungal infections. PMNs possess a diverse arsenal of components to combat microorganisms, including the production of reactive oxygen species and release of degradative enzymes and antimicrobial peptides. Despite numerous PMNs at the site of gonococcal infection, N. gonorrhoeae can be cultured from the PMN-rich exudates of individuals with acute gonorrhea, indicating that some bacteria resist killing by neutrophils. The contribution of PMNs to gonorrheal pathogenesis has been modeled in vivo by human male urethral challenge and murine female genital inoculation and in vitro using isolated primary PMNs or PMN-derived cell lines. These systems reveal that some gonococci survive and replicate within PMNs and suggest that gonococci defend themselves against PMNs in two ways: they express virulence factors that defend against PMNs' oxidative and non-oxidative antimicrobial components, and they modulate the ability of PMNs to phagocytose gonococci and to release antimicrobial components. In this review, we will highlight the varied and complementary approaches used by N. gonorrhoeae to resist clearance by human PMNs, with an emphasis on gonococcal gene products that modulate bacterial-PMN interactions. Understanding how some gonococci survive exposure to PMNs will help guide future initiatives for combating gonorrheal disease.

Resistance of Neisseria gonorrhoeae to neutrophils.

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that are exquisitely adapted for growth at human mucosal surfaces and for efficient transmission between hosts. One factor that is essential to neisserial pathogenesis is the interaction between the bacteria and neutrophils, which are recruited in high numbers during infection. Although this vigorous host response could simply reflect effective immune recognition of the bacteria, there is mounting evidence that in fact these obligate human pathogens manipulate the innate immune response to promote infectious processes. This Review summarizes the mechanisms used by pathogenic neisseriae to resist and modulate the antimicrobial activities of neutrophils. It also details some of the major outstanding questions about the Neisseria-neutrophil relationship and proposes potential benefits of this relationship for the pathogen.

A bacterial siren song: intimate interactions between Neisseria and neutrophils.

Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome.

Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils.

The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniformly Opa negative (Opa(-)), whereas cultures of predominantly Opa(-) parental Gc and an intermediate lacking the "translucent" subset of opa genes (Î”opaBEGK) stochastically gave rise to Opa-positive (Opa(+)) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa(-) bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD(+) Gc survived less well after exposure to PMNs compared to Opa(-) bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology.

Constitutively Opa-expressing and Opa-deficient neisseria gonorrhoeae strains differentially stimulate and survive exposure to human neutrophils.

The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae.

Neisseria gonorrhoeae metalloprotease NGO1686 is required for full piliation, and piliation is required for resistance to H2O2- and neutrophil-mediated killing.

Article	Year
Coordinate regulation of Salmonella enterica serovar Typhimurium invasion of epithelial cells by the Arp2/3 complex and Rho GTPases. Infection and immunity\| PubMed ID: 12704163	2003
The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae. Molecular microbiology\| PubMed ID: 16194236	2005
The transcriptome response of Neisseria gonorrhoeae to hydrogen peroxide reveals genes with previously uncharacterized roles in oxidative damage protection. Molecular microbiology\| PubMed ID: 16194237	2005
Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili. Cellular microbiology\| PubMed ID: 16922862	2006
Transposon mutagenesis identifies sites upstream of the Neisseria gonorrhoeae pilE gene that modulate pilin antigenic variation. Journal of bacteriology\| PubMed ID: 17307859	2007
Neisseria gonorrhoeae suppresses the oxidative burst of human polymorphonuclear leukocytes. Cellular microbiology\| PubMed ID: 18684112	2008
Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes. Cellular microbiology\| PubMed ID: 19290914	2009
Mismatch correction modulates mutation frequency and pilus phase and antigenic variation in Neisseria gonorrhoeae. Journal of bacteriology\| PubMed ID: 19854909	2010
Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae. Journal of bacteriology\| PubMed ID: 19933360	2010
The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation. Molecular microbiology\| PubMed ID: 21299643	2011
Resistance of Neisseria gonorrhoeae to neutrophils. Frontiers in microbiology\| PubMed ID: 21747795	2011
A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nature reviews. Microbiology\| PubMed ID: 22290508	2012
Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils. Cellular microbiology\| PubMed ID: 23374609	2013
Constitutively Opa-expressing and Opa-deficient neisseria gonorrhoeae strains differentially stimulate and survive exposure to human neutrophils. Journal of bacteriology\| PubMed ID: 23625842	2013
Neisseria gonorrhoeae metalloprotease NGO1686 is required for full piliation, and piliation is required for resistance to H2O2- and neutrophil-mediated killing. mBio\| PubMed ID: 23839218	2013

Alison K. Criss

Biography

JoVE Journal Publications

Other Publications