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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Infect Dis. Author manuscript; available in PMC 2010 August 15.
Published in final edited form as:
PMCID: PMC2768539

Haemophilus ducreyi Activates Human NK Cells through Interactions with Macrophages and Dendritic Cells


The role of natural killer (NK) cells in the host response to Haemophilus ducreyi is unclear. In pustules obtained from infected human volunteers, there was an enrichment of CD56bright NK cells bearing the activation markers CD69 and HLA-DR compared to peripheral blood. To study the mechanism by which H. ducreyi activated NK cells, we used peripheral blood mononuclear cells from uninfected volunteers. H. ducreyi activated NK cells only in the presence of antigen presenting cells. H. ducreyi-infected monocytes and monocyte-derived macrophages activated NK cells in a contact- and IL-18-dependent manner, while monocyte-derived dendritic cells induced NK activation through soluble IL-12. More lesional NK cells produced IFN-γ in response to IL-12 and IL-18 than peripheral blood NK cells. We conclude that NK cells are recruited to experimental lesions and likely are activated by infected macrophages and dendritic cells. IFN-γ produced by lesional NK cells may facilitate phagocytosis of H. ducreyi.

Keywords: Haemophilus ducreyi, chancroid, human trials, NK cells, antigen presenting cells, IFN-γ, monokines

Haemophilus ducreyi causes chancroid, which facilitates the transmission of HIV-1 [1]. To understand the immunopathogenesis of chancroid, we developed a human challenge model, in which healthy adults are infected with H. ducreyi on the upper arm [2, 3]. Papules form within 24 hours of inoculation and either spontaneously resolve or evolve into pustules within 2 to 5 days. The cutaneous immune response in experimental infection consists of neutrophils, macrophages, T cells and myeloid dendritic cells (DC) [3, 4]. In pustules, H. ducreyi is surrounded by neutrophils and macrophages, which fail to ingest the organism [5, 6]. Similar relationships between H. ducreyi and host cells are found in chancroidal ulcers [7]. Thus, the model is relevant to natural disease.

Natural killer (NK) cells constitute a first line of defense against infection. NK cells modulate both innate and adaptive immunity through their ability to secrete chemokines and cytokines, such as IFN-γ[8]. Human NK cells are defined as CD56+ and CD3. Approximately 90% of circulating NK cells are CD56dim, while the remainder are CD56bright [9]. CD56bright cells are potent cytokine producers with limited cytotoxic activity; CD56dim cells have potent cytotoxic capacity with lower ability to produce cytokines [9]. NK cell activity is controlled by the balance between the inhibitory and activating signals mediated through several receptors and co-stimulatory molecules [10] and by cytokines such as IL-12, IL-18, IL-10, and TGF-β [11]. Pathogens may activate NK cells directly or by interactions with antigen presenting cells (APC) such as monocytes, macrophages or DC [11].

We had reported that NK cells comprise only 1% of the mononuclear cell infiltrate in experimental chancroid by immunostaining with antibodies to CD57 [12]. CD57 is expressed by most CD56dim cells but not by CD56bright cells [13]. Here we reevaluated the role of NK cells in the host response to H. ducreyi. We studied the mechanism(s) by which H. ducreyi activated NK cells in vitro and determined the functional properties of NK cells in infected tissues.


Bacterial strains and growth conditions

H. ducreyi strain 35000HP (HP, human passaged) was described previously [14]. Bacteria were grown on chocolate agar plates and GC medium broth as described [15]. Heat-killed bacteria were prepared by incubation at 65°C for 1 hour.

Human subjects

Fourteen healthy adults, who were infected with 35000HP alone (this study) or 35000HP and isogenic mutants of 35000HP in several mutant vs. parent comparison trials until they developed pustules, contributed skin biopsies (table 1). Since the immunopathology caused by mutant strains that form pustules is identical to that by caused by 35000HP [16], we analyzed biopsies obtained from parent and mutant-infected sites. Informed consent was obtained in compliance with the human experimental guidelines of the US Department of Health and Human Services and the Institutional Review Board of Indiana University-Purdue University at Indianapolis. Enrollment and exclusion criteria, preparation of H. ducreyi and inoculation procedures were reported previously [14]. Single cell suspensions were obtained from biopsies as described [4]. Peripheral blood mononuclear cells (PBMC) were also obtained on the day of biopsy from each subject as described [4]

Sources of tissue samples

Characterization of NK cells in lesions and blood of infected subjects

Cells from biopsies and PBMC were incubated with allophycocyanin conjugated antibodies to CD3, phycoerythrin (PE) conjugated anti-CD56 antibodies, peridinin chlorophyll protein (PerCP) conjugated anti-HLA-DR antibodies, and isothiocyanate (FITC) conjugated anti-CD 69 antibodies (BD Biosciences). Samples were washed, fixed with 2% paraformaldehyde in PBS, and analyzed using a FACSCalibur flow cytometer and the BD CellQuest Pro version 4.0.1 software (BD Biosciences). The proportion of NK cells was calculated among lymphocytes, identified by their forward and side scatter properties. Ten thousand lymphocytes were analyzed for PBMC samples; all collected lymphocytes (range, 770 to 23,000 cells) were analyzed from the biopsies.

To detect intracellular IFN-γ production, paired biopsy and PBMC samples were incubated in growth medium alone (RPMI 1640, 10% heat inactivated FBS, 55 µM 2-mercaptoethanol, and 2 mM glutamine) or in medium supplemented with 10 ng/ml of both recombinant human IL-12 (eBiosciences) and IL-18 (R&D Systems) for 18 hours at 37°C and 5% CO2. Monensin (Sigma-Aldrich) was added to the culture at a final concentration of 3 µM. Cells were collected, stained with anti-CD3-allophycocyanin and anti-CD56-PE antibodies, fixed, permeabilized with 0.2% saponin (Sigma-Aldrich) and stained with anti-IFN-γ−FITC antibodies (eBiosciences).

Examination of H. ducreyi activation of NK cells using cells derived from blood of uninfected donors

PMBC were isolated from leukopacks purchased from the Central Indiana Regional Blood Center from 15 anonymous donors. CD14+ monocytes were purified by positive selection using magnetic CD14 microbeads (Miltenyi Biotech). The CD14 fraction was used to isolate NK cells by negative selection using the Human NK Isolation Kit (Miltenyi Biotech). To generate monocyte-derived macrophages (MDM), CD14+ monocytes were cultured in growth medium for 6 days at 37°C and 5% CO2. Recombinant human macrophage-colony stimulating factor (R&D Systems) (5 ng/ml) was added at day 0, 2, and 4. Monocyte-derived DC were differentiated from CD14+ cells as described [4], except that recombinant human granulocyte macrophage-colony stimulating factor and IL-4 were each used at a concentration of 5 ng/ml.

Heat-killed or live bacteria were centrifuged onto wells containing PBMC. After 90 minutes of incubation at 35°C, cells were incubated at 37°C for an additional 5.5 h. Monensin was added to the culture at a concentration of 3 µM in the last 3 h of incubation. Cells were stained with anti-CD3-allophycocyanin, anti-CD56-PE and anti-CD69-FITC or with anti-CD3-allophycocyanin, anti-CD56-PE and anti-IFN-γ-FITC. Optimal production of IFN-γ occurred with heat-killed H. ducreyi at a ratio of 10:1 and with live H. ducreyi at a ratio of 1:1. Higher ratios of live bacteria may have inhibited NK cell activation by production of cytolethal distending toxin, which kills lymphocytes [17]. These parameters were used throughout the remainder of the experiments.

CD14+ monocytes and MDM were mixed with autologous NK cells at a ratio of 1:1 and 1:3, respectively, and incubated with media, heat-killed or live H. ducreyi as described above. Gated NK cells were analyzed for surface expression of CD69 and intracellular IFN-γ production.

DC were incubated with media, heat-killed or live H. ducreyi for 6 hours and treated with 100 µg/ml of gentamicin for 30 minutes to kill extracellular H. ducreyi, washed three times and suspended in growth medium. Approximately 1 × 105 DC were co-cultured with 3 × 105 autologous NK cells for an additional 24 hours in the presence of 100 U/ml penicillin and 100 µg/ml streptomycin. Gated NK cells were analyzed for surface expression of CD69. Culture supernatants were assayed for IFN-γ and IL-12p70 production using enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences).

For transwell experiments, the APC infected with live H. ducreyi were seeded in 24-well tissue culture plates. An insert with a 0.4-µm membrane (Corning) was placed in the well, and purified NK cells were added to the top chamber. For antibody blocking experiments, the APC were incubated with 20 µg/ml of neutralizing anti-IL-12p40 (BD Biosciences), anti-IL-18 (R&D Systems), or mIgG1 isotype control antibodies (eBiosciences) for 45 minutes at 4°C before co-culture with NK cells or NK cells were preincubated with 20 µg/ml anti-NKG2D and anti-2B4 antibodies for 45 minutes at 4°C.

Statistical analysis

Paired t-tests were used to analyze the data, and nominal P values are reported for each paired comparison; P values ≤ 0.05 were considered significant. Due to the sample size, we did not adjust for multiple comparisons.


NK cells are recruited to H. ducreyi-infected skin

We examined whether NK cells were recruited to pustules induced by experimental infection with H. ducreyi. Biopsies and PBMC obtained from 14 subjects experimentally infected for a mean ± SD of 7.1 ± 0.8 days were analyzed by flow cytometry. The ratio of CD56+CD3 cells to all lymphocytes was compared between the lesional cells and PBMC. There was no significant difference in the percentage of NK cells recruited to lesions versus PBMC (mean ± SD, 12.0 ± 1.5% and 10.4 ± 0.9%, respectively, P = 0.3).

To determine whether a subset of NK cells were expanded in infected skin, cells from 11 paired samples of PBMC and pustules were compared for surface density of CD56. A representative result is shown in figure 1A. The majority of NK cells in PMBC were CD56dim. Compared to PBMC, there was a statistically significant enrichment of CD56bright cells in pustules (figure 1B). The percentages of CD69+ and HLA-DR+ NK cells at infected sites were also significantly higher than in PBMC, indicating that lesional NK cells were activated (figure 1B).

Figure 1
Characterization of the NK cell infiltrates at H. ducreyi-infected sites. A. FACS dot plots of NK cells in PBMC and a pustule obtained simultaneously from 1 subject experimentally infected with H. ducreyi. The numbers in the dot plots represent the percentages ...

H. ducreyi activates NK cells indirectly

We sought to identify the mechanisms by which NK cells were activated by H. ducreyi. Since pustules yield too few cells for such studies, we used PBMC from anonymous blood donors as surrogates for lesional cells. We depleted PBMC of CD14+ monocytes and stimulated them with heat-killed or live bacteria. CD14 PBMC failed to promote IFN-γ production by NK cells in comparison to PBMC (figure 2A). Infection of purified NK cells with H. ducreyi did not induce appreciable amounts of IFN-γ (data not shown). Therefore, H. ducreyi did not activate NK cells directly, and APC such as the CD14+ monocytes were involved in H. ducreyi-induced NK cell activation.

Figure 2
CD14+ monocyte-mediated NK cell activation. A. PBMC or CD14-depleted PBMC were stimulated with heat-killed (HK) or live bacteria. Incubation in medium (med) served as a negative control. The values represent the mean ± SD of IFN-γ+ NK ...

H. ducreyi-infected CD14+ monocytes and macrophages activate NK cells by contact-dependent mechanisms

To identity how CD14+ cells activated NK cells, we co-cultured infected CD14+ monocytes and autologous NK cells with and without transwell inserts. H. ducreyi infection of CD14+ cells promoted NK cell production of IFN-γ and upregulation of CD69 (figure 2B, 2C). Treatment of the CD14+ cells with cytochalasin D (5–20 µM) abrogated subsequent NK cell activation (data not shown). IFN-γ production and CD69 expression by NK cells were completely blocked by separating H. ducreyi infected CD14+ cells from NK cells (figure 2B, 2C), indicating a crucial role of contact-dependent signals.

APC and NK cells form conjugates during co-cultures, and targeted secretion of IL-12 and IL-18 by APC to the conjugate interface promotes NK cell activation [1820]. Therefore, we investigated the role of IL-12 and IL-18 in CD14+ dependent NK cell activation. Blocking with anti-IL-12 neutralizing antibodies had no effect on NK cell activation, whereas anti-IL-18 neutralizing antibodies significantly reduced the ability of NK cells to make IFN-γ or to upregulate CD69 (figure 2B, 2C). IL-18 was produced by CD14+ monocytes in response to heat-killed and live H. ducreyi (figure 2D); IL-12 was undetectable in these culture supernatants (data not shown). Addition of IL-18 at up to 100 ng/ml did not rescue IFN-γ production by NK cells in transwell experiments (data not shown), suggesting that factors other than IL-18 were involved in contact-dependent NK cell activation.

Receptor/ligand interactions between APC and NK cells contribute to NK cell activation [11]. For example, the triggering of the NK receptor NKG2D or ligation of the NK costimulatory molecule 2B4 promotes IFN-γ production [21, 22]. To determine whether these receptors were involved in H. ducreyi-induced activation, neutralizing antibodies against NKG2D and 2B4 were added to infected CD14+ monocyte and NK cell co-cultures. Blocking NKG2D and 2B4 had no effect on the ability of NK cells to produce IFN-γ or to upregulate CD69 (figure 2B, 2C). Interactions between CD40, CD80, and CD86 on monocytes/macrophages with their ligands on NK cells can also provide contact-dependent signals for NK cell activation [11, 23, 24]. Neutralizing antibodies to CD40L, CD80, and CD86 also did not prevent NK cell activation (data not shown). Thus, cell surface receptors NKG2D, 2B4, CD40, CD80, and CD86 were not involved in the contact dependent activation of NK cells by CD14+ cells.

In pustules, macrophages comprise approximately 11.5 % of the mononuclear cell infiltrates [12]. Thus, we co-cultured MDM with NK cells. NK cells produced IFN-γ and upregulated CD69 in response to H. ducreyi in the presence of MDM (figure 3A, 3B). MDM-mediated NK cell activation was blocked by transwell separation and by anti-IL-18 antibodies but not by anti-NKG2D, anti-2B4, or anti-IL-12 neutralizing antibodies (figure 3A, 3B). Taken together, the data suggest that phagocytosis, unidentified cell-contact signal(s) and IL-18 produced by infected CD14+ monocytes/macrophages were required for CD14+ cells/macrophages to activate NK cells in response to H. ducreyi.

Figure 3
Contact- and IL-18-dependent activation of NK cells by MDM in co-cultures. MDM and NK cells were incubated in medium (med) or stimulated with heat-killed (HK) or live H. ducreyi together in the presence of isotype control antibodies (iso), anti-IL-12, ...

Cell contact-independent and IL-12 dependent activation of NK cells by H. ducreyi-infected DC

Myeloid DC are recruited to H. ducreyi-infected sites and produce proinflammatory cytokines, including IL-6 and IL-12 [4, 25]. We tested H. ducreyi-infected myeloid DC for their capacity to stimulate NK cells. The monocyte-derived DC were infected with H. ducreyi for 6 hours and subsequently co-cultured with autologous NK cells. Under these conditions, both heat-killed and live H. ducreyi were able to induce IFN-γ production by NK cells (figure 4A). Transwell separation did not prevent IFN-γ production, suggesting that secreted factors were sufficient to activate NK cells (figure 4A). Since IL-12 secreted by maturing DC is a potent activator of IFN-γ production by NK cells [2628], we investigated the role of IL-12 in NK cell activation. Blocking with anti-IL-12 antibody significantly reduced IFN-γ production by NK cells (figure 4A). In contrast, anti-IL-18 antibodies had no effect on DC-induced IFN-γ production by NK cells (data not shown).

Figure 4
NK cell activation by H. ducreyi-infected DC. NK cells were co-cultured with DC that had been incubated in medium (med) or activated with heat-killed (HK) or live H. ducreyi in the presence of isotype control antibodies (iso), anti-IL-12 antibodies, or ...

DC activated with H. ducreyi upregulated CD69 expression by NK cells. Separating DC from NK cells by transwell inserts slightly but significantly reduced CD69 expression. Anti-IL-12 and anti-IL-18 antibodies had little effect on CD69 upregulation (figure 4B and data not shown). Therefore, IFN-γ production and CD69 expression by NK cells were differentially regulated by DC-derived signals in response to H. ducreyi.

Activated NK cells can induce expression of IL-12 and surface activation markers by DC [27, 29, 30]. We measured IL-12 production by H. ducreyi-infected DC in the presence or absence of activated NK cells. We did not observe significant enhancement of IL-12 production by DC and NK cell co-cultures compared to DC alone in response to heat-killed or live H. ducreyi (figure 4C). In addition, NK cells did not increase the expression of HLA-DR, CD40, CD80, CD83 and CD86 on DC (data not shown).

Lesional NK cells produce IFN-γ

Since IL-12 and IL-18 produced by H. ducreyi-infected DC and monocytes/macrophages induced IFN-γ production by NK cells in vitro, we analyzed the role of these monokines in stimulating NK cells from H. ducreyi-infected tissues to produce IFN-γ. Cells from six pustules paired with autologous PBMC were cultured in medium with or without both IL-12 and IL-18. Representative results obtained from one subject are shown in figure 5A. NK cells from lesions or PBMC made little IFN-γ in the absence of monokines. Addition of exogenous IL-12 and IL-18 induced IFN-γ production by lesional as well as peripheral blood NK cells. Compared with peripheral blood NK cells, significantly more lesional NK cells made IFN-γ (figure 5B), suggesting that lesional NK cells have the capacity to make IFN-γ in vivo.

Figure 5
Expression of IFN-γ by peripheral blood and lesional NK cells in response to IL-12 and IL-18 stimulation. A. FACS dot plots of IFN-γ production by NK cells from a pair of peripheral blood and pustule samples. Incubation in medium (med) ...


We previously reported that CD57+ NK cells are a minor component of the cutaneous immune response to experimental H. ducreyi infection [12]. However, recent studies have demonstrated that the CD56bright NK subset, which is CD57 [13], is highly expanded at sites of inflammation [3133]. Although the proportion of NK cells was not enriched in infected tissue versus peripheral blood, there was a significant enrichment of CD56bright NK cells in experimental lesions.

The enrichment of CD56bright NK cells could result from their preferential recruitment to H. ducreyi-infected skin. CD56bright cells utilize CCR5, CCR2 or CXCR3 to enter inflamed sites [9, 33, 34], while CD56dim cells migrate to inflamed tissues through CXCR1, CX3CR1 and ChemR23 [9, 3436]. Transcripts for several chemokines that attract CD56bright cells, such as the CXCR3 ligands, CXCL9, CXCL10 and CXCL11, are highly expressed in H. ducreyi-infected tissues compared to PBS-inoculated skin [25]. Transcripts for CCR2 and CCR5 ligands, including CCL2, CCL4, CCL5, CCL7 and CCL8, are also upregulated [25]. H. ducreyi-infected skin also has increased levels of transcripts for the CXCR1 and CX3CR1 ligands IL-8 and CX3CL1 [25]. Therefore, both CD56bright and CD56dim NK subsets could be recruited to infected skin; the enrichment of CD56bright cells might reflect their preferential migration towards high concentrations of CXCL9, CXCL10 and CXCL11 produced at lesional sites. Alternatively, CD56dim cells could be converted to CD56bright cells by the action of cytokines and other factors found in infected microenvironments, such as IL-2, IL-12 and IL-15 or IFNα [31, 37]. CD56bright cells might have survival or proliferative advantages over CD56dim cells, as has been reported [26, 38]. Further study is required to define the mechanisms by which CD56bright cells accumulate in H. ducreyi-infected skin.

CD14+ monocytes, MDM, or DC induced peripheral blood NK cells to express IFN-γ and to upregulate CD69 in response to H. ducreyi in vitro. NK cell activation by monocytes/macrophages required cell-to-cell contact and IL-18. H. ducreyi-infected monocytes produced IL-18 in a picogram per milliliter range, and direct contact between monocytes and NK cells may be required for IL-18 function [18, 20]. Higher concentrations of exogenous IL-18 did not restore IFN-γ production when infected CD14+ and NK cells were physically separated, consistent with the finding that IL-18 alone is not able to activate NK cells [18, 39]. We found no evidence that IL-12, the engagement of the NK activating receptors NKG2D and 2B4 or the costimulatory molecules CD40, CD80, and CD86 had a role in monocytes/macrophage-mediated NK cell activation by H. ducreyi. It is possible that IFN-α/β [11, 40] or other contact-dependent signals may synergize with IL-18 to promote NK cell activation by H. ducreyi.

Surprisingly, NK cell activation by monocytes was inhibited by cytochalasin D. Although H. ducreyi is relatively resistant to phagocytosis, 5 to 10% of monocytes and macrophages ingest the organism in vitro [41, 42]. Similarly, phagocytosis of Borrelia burgdorferi and Treponema pallidum by PMBC is required to promote IFN-γ production by NK cells[43].

Blocking with anti-IL-12p40 antibody significantly reduced IFN-γ production by NK cells co-cultured with H. ducreyi-infected DC. Anti-IL-12p40 inhibits the function of both IL-12 and IL-23, but H. ducreyi-infected DC did not produce IL-23 as measured by ELISA (data not shown). In addition, IL-23 alone does not activate NK cells [44], Thus, IL-12 was likely critical for DC-mediated NK cell activation.

The IFN-γ response of NK cells to H. ducreyi-infected DC was mediated by IL-12 in a contact-independent manner. Previously, we reported that DC infected with live H. ducreyi produced nanogram levels of IL-12, much greater than those secreted by LPS-stimulated DC [4]. Thus, it is plausible that large amounts of IL-12 produced by H. ducreyi-infected DC were sufficient to promote IFN-γ production by NK cells. .

A significant portion of NK cells in pustules carried the activation markers CD69 and HLA-DR, indicating that they were activated. Lesional NK cells had an enhanced capacity to produce IFN-γ in response IL-12 and IL-18, compared to peripheral blood NK cells. Similar sensitivity to monokine stimulation has been observed for NK cells from chronic inflammatory sites [31, 32] and may reflect priming and/or activation of lesional NK cells by monokines or other factors within infected tissue. Upregulation of monokine receptors by IL-12 or IFN-α [39, 45] may allow NK cells to respond more vigorously to low concentrations of monokines.

This study was limited to characterization of NK cells that were recruited to pustules. However, host responses to experimental H. ducreyi infection may lead to pustule formation or spontaneous resolution of infection [5, 6]. The basis for disease resolution is unknown, but likely is due to the ability of phagocytes to clear the bacteria. When challenged twice with H. ducreyi, volunteers tend to resolve infection twice (RR group), or form pustules twice (PP group), indicating that different hosts are differentially susceptible to infection [25]. Microarray analysis of infected skin obtained from PP and RR subjects challenged a third time shows that the groups share a core transcript response to H. ducreyi [25]. Transcripts that signify an effective immune response are exclusively expressed in RR infected skin, while those consistent with a hyperinflammatory, dysregulated response are expressed in PP infected skin [25]. The microenvironment at infected sites, including the balance between activating and inhibitory signals, could shape NK or T cell responses that modulate phagocytosis and contribute to clearance of H. ducreyi or disease progression. We are currently investigating whether there are any differences in NK recruitment and function and how DC and other APCs differentially regulate NK cells in the RR and PP groups.

Interestingly, monocyte-derived DC from the RR group have a transcript response to H. ducreyi that is typical of type 1 DC response, while those from the PP group have a mixed type 1 and regulatory DC response [25]. In the RR group, DC might direct NK or T cells to make an appropriate amount of IFN-γ during the early phase of infection, which augments phagocytosis and bactericidal activity by neutrophils and macrophages. Alternatively, excess IFN-γ production by NK or T cells might promote hyperinflammatory and dysregulated immune responses seen in pustule formers [25]. We noted marked variation in IFNγ production by NK cells in the PBMC obtained from the uninfected anonymous donors. We plan to also determine whether donor variations in IFN-γ production by NK cells are associated with resistance or susceptibility to H. ducreyi infection in humans.


We thank Drs. Byron E. Batteiger, Margret E. Bauer, David S. Wilkes and Barbara Van Der Pol for their critical review of the manuscript and the volunteers for their participation in these studies.

Financial Support: This work was supported by grants AI31494 and AI059384 from the National Institute of Allergy and Infectious diseases. The human challenge trials were supported by grant MO1RR00750 to the General Clinic Research Center at Indiana University.


Potential conflicts of interest: no conflicts.

Presented in part: 2008 Keystone Symposia: NK and NKT Cell Biology and Innate Immunity, Keystone, CO, February 24–29, 2008 (abstract 241); 108th General Meeting of the American Society for Microbiology, Boston, MA, June 1–5, 2008 (abstract E052).


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