Effect of inflammatory response on in vivo competition between two chlamydial variants in the guinea pig model of inclusion conjunctivitis

Abstract

In order to study the interaction of variants in in vivo infection, we employed an azithromycin-resistant mutant (AZ(2)) and its wild-type parent (SP(6)) in the guinea pig model of Chlamydia caviae conjunctival infection. When each strain was inoculated individually into conjunctiva, both attained the same level of growth, but AZ(2) elicited less pathology. However, when equal numbers of the two strains were inoculated together into the guinea pig conjunctiva, SP(6) produced a significantly greater number of inclusion-forming units than AZ(2), and the pathology reflected that of a SP(6) monoinfection. The goal of this study was to further characterize the dynamics of concomitant infection of these two distinct variants, with particular emphasis on the impact of the host response on the in vivo growth of each organism and the development of pathology. Animals infected with AZ(2) had reduced conjunctival infiltration with CD45(+) cells and neutrophils as well as a reduced interleukin-8 (IL-8) response. Gene expression of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), CCL2, and CCL5 was also significantly lower in AZ(2)-infected animals. The lower inflammatory response induced by AZ(2) was associated with its decreased ability to activate NF-κB via Toll-like receptor 2 (TLR2). In general, the inflammatory response in animals infected with both variants was greater than in infection with AZ(2) alone, resulting in lower numbers of AZ(2) than those of SP(6) in the mixed infection. Our results suggest that the ability to elicit an inflammatory response is an important factor in the dynamics of mixed infection with strains that display different pathological phenotypes.

Fig 1

Effect of various doses of SP₆, AZ₂, and mixed SP₆ and AZ₂ inocula on the gross pathological response in the conjunctiva. The curves represent repeated daily scores of five animals in each group.

Fig 2

Kinetics of bacterial growth following conjunctival infection with various doses of SP₆, AZ₂, and mixed SP₆ and AZ₂. The first column represents the growth curves of chlamydiae in animals infected with SP₆ and AZ₂ individually and the total number of IFU of the mixed infection. The second column represents the SP₆ and AZ₂ populations within the mixed infection. The curves represent repeated numbers of IFU for five animals in each group. The statistical analysis was performed using a 2-way ANOVA on repeated measures with a Tukey post hoc analysis. The mean competitive index (CI) for the mixed infection is shown at day 6, the time of the peak infection and maximum IFU difference between the two variants in the second column. The CI represents the ratio of the output AZ₂/SP₆ on day 6 compared to the input AZ₂/SP₆ ratio. The input dose was a ratio of 1:1.

Fig 3

CD45⁺ and PMN levels in conjunctival tissue collected at various times after infection with SP₆, AZ₂, or SP₆ and AZ₂ mixed. Each point represents the mean of four animals. The cell numbers are the total number of each cell type in the entire upper conjunctiva of one eye. The mean pathology scores assessed in all animals on the days prior to and at euthanasia are included in the top panel for comparison to the cell numbers. The statistical analysis was performed using a 2-way ANOVA with a Tukey post hoc analysis.

Fig 4

IL-8 levels in conjunctival secretions collected at the time of euthanasia. Each point represents the mean of four animals. The mean pathology scores assessed in all animals on the days prior to and at euthanasia are included in the top panel for comparison to the IL-8 levels. The statistical analysis was performed using a 2-way ANOVA with a Tukey post hoc analysis.

Fig 5

Decreased NF-κB activity in vitro in HEK293-hTLR2 cells infected with AZ₂ or SP₆ at 6 and 24 h postinoculation. The supernatants from the 6-h cultures were undiluted, while those from the 24-h cultures were diluted 10-fold so that the values would be within the range of the standards. The bars represent the means and standard deviations of triplicate cultures. Pam3CSK4 (PAM) served as the positive control. The statistical analysis was performed using a 2-way ANOVA with a Tukey post hoc analysis. The P values represent the comparison of the AZ₂ and SP₆ variants at each MOI. OD₄₀₅, optical density at 405 nm.