The Cryptic Plasmid Improves Chlamydia Fitness in Different Regions of the Gastrointestinal Tract

Abstract

The cryptic plasmid is important for chlamydial colonization in the gastrointestinal tract. We used a combination of intragastric, intrajejunal, and intracolon inoculations to reveal the impact of the plasmid on chlamydial colonization in distinct regions of gastrointestinal tract. Following an intragastric inoculation, the plasmid significantly improved chlamydial colonization. At the tissue level, plasmid-positive Chlamydia produced infectious progenies throughout gastrointestinal tract. However, to our surprise, plasmid-deficient Chlamydia failed to produce infectious progenies in small intestine, although infectious progenies were eventually detected in large intestine, indicating a critical role of the plasmid in chlamydial differentiation into infectious particles in small intestine. The noninfectious status may represent persistent infection, since Chlamydia genomes proliferated in the same tissues. Following an intrajejunal inoculation that bypasses the gastric barrier, plasmid-deficient Chlamydia produced infectious progenies in small intestine but was 530-fold less infectious than plasmid-positive Chlamydia, suggesting that (i) the noninfectious status developed after intragastric inoculation might be induced by a combination of gastric and intestinal effectors and (ii) chlamydial colonization in small intestine was highly dependent on plasmid. Finally, following an intracolon inoculation, the dependence of chlamydial colonization on plasmid increased over time. Thus, we have demonstrated that the plasmid may be able to improve chlamydial fitness in different gut regions via different mechanisms, which has laid a foundation to further reveal the specific mechanisms.

Keywords: Chlamydia; Chlamydia muridarum; GI tract colonization; gastrointestinal tract; persistence; plasmid.

FIG 1

Comparison of live organism shedding between mice infected intragastrically with Chlamydia muridarum with or without plasmid. Groups of C57BL/6J (C57) mice (n = 4 to 6) inoculated intragastrically with C. muridarum with (plasmid positive) (a to d) or without (plasmid deficient) (e to h) plasmid were monitored for live chlamydial organism shedding in rectal swabs on days 3 and 7 and weekly thereafter, as indicated along the x axis. Different inoculation doses ranging from 10³ to 10⁶ inclusion forming units (IFU) per mouse were used for different groups of mice as indicated on the right, while the live organisms recovered from rectal swabs are expressed as log₁₀ IFU as shown along the left y axis.

FIG 2

Comparison of live organism recovery from gastrointestinal tissues between mice inoculated intragastrically with Chlamydia muridarum with or without plasmid. Groups of C57 mice (n = 3 to 5) intragastrically inoculated with 1 × 10⁵ IFU of Chlamydia muridarum with (plasmid positive) (a to e) or without (plasmid deficient) (f to j) plasmid were sacrificed to quantitate live chlamydial organism recovery in different regions of gastrointestinal tract as shown along the x axis on days 3 and 7 and weekly thereafter, as indicated on the right. The live organisms recovered from different tissues are expressed as log₁₀ IFU as shown along the left y axis.

FIG 3

Chlamydial genome copies recovered from gastrointestinal tissues of mice inoculated intragastrically with plasmid-deficient Chlamydia. Groups of C57 mice (n = 3 to 5) intragastrically inoculated with 1 × 10⁵ IFU of plasmid-deficient Chlamydia muridarum (as described in the legend for Fig. 2) were sacrificed to quantitate chlamydial genome copies in different regions of gastrointestinal tract as shown along the x axis on days 3 (a) and 7 (b) and weekly thereafter (c to e), as indicated on the right. The genome copies recovered from different tissues are expressed as log₁₀ genomes per tissue as shown along the y axis.

FIG 4

Comparison of live organism recovery from gastrointestinal tissues between mice infected via intrajejunal inoculation with Chlamydia muridarum with or without plasmid. Groups of C57 mice (n = 3 to 5) infected via intrajejunal inoculation with different doses (as shown in individual panels) of C. muridarum with (plasmid positive) (a to d) or without (plasmid free) (e to h) plasmid were sacrificed to quantitate live chlamydial organism recoveries in different regions of gastrointestinal tract as shown along the x axis on day 3 after intrajejunal inoculation. The live organisms recovered from different tissues are expressed as log₁₀ IFU as shown along the left y axis. The doses required for infecting 50% of mice (ID₅₀) were calculated for samples collected from jejunum and colon and are listed at the bottom (below the corresponding samples).

FIG 5

Comparison of live organism shedding between mice infected via intracolon inoculation with Chlamydia muridarum with or without plasmid. Groups of C57 mice (n = 4 to 6) inoculated via intracolon inoculation with 1 × 10⁵ IFU of C. muridarum with (plasmid positive) (a) or without (plasmid deficient) (b) plasmid were monitored for live chlamydial organism shedding in rectal swabs on days 3 and 7 and weekly thereafter, as indicated along x axis. The live organisms recovered from rectal swabs are expressed as log₁₀ IFU, as shown along the y axis. The level of colonization by plasmid-deficient Chlamydia was significantly lower than that of plasmid-positive Chlamydia (area under curve, Wilcoxon rank sum, P < 0.05).

FIG 6

Comparison of live organism recovery from rectal swabs between mice infected via intracolon inoculation with Chlamydia muridarum with or without plasmid. Groups of C57 mice (n = 3 to 5) infected via intracolon inoculation with different doses (as shown in individual panels) of C. muridarum with (plasmid positive) (a to d) or without (plasmid deficient) (e to h) plasmid were monitored for live chlamydial organism recovery in rectal swabs on days 3 and 7 and weekly thereafter after intracolon inoculation (as shown along x axis). The live organisms recovered are expressed as log₁₀ IFU, as shown along the y axis. The doses required for infecting 50% of the mice (ID₅₀) were calculated for samples collected at individual time points and are listed at the bottom (below the corresponding time points).