The predicted proteins encoded within these regions of the 3 parents and 12 recombinants were then compared using the MUSCLE sequence alignment software, and a total of 124 proteins had at least one non-synonymous amino acid change that was associated with the attachment phenotype (Additional file 2: Table S1). The chlamydial membrane proteins PmpE (14 amino acid changes), PmpF (110 AA changes), PmpG (28 AA changes), and PmpH (57 AA changes) were among the proteins

with the highest number of non-synonymous amino acid changes. Other relevant genes that were associated with high attachment efficiency were ORFs CT089, and CT860 – 862, ORFs encoding proteins involved in the Type III secretion process [28, 29]. Differences in the sequences of proteins demonstrated selleck compound by GSI-IX others to function in primary attachment (OmpA, [30], OmcB [31]) or proposed to be associated with very early events following contact (HSP70, [32]) were not associated with differential attachment efficiency, as measured by our assay (Figure 6). Variation in secondary inclusion formation between find more Recombinant strains Formation of secondary inclusions in infected cells is another trait that varies among strains and serovars. For example, strains of serovars G and F commonly form secondary inclusions at a higher rate than strains of serovar J and L2 [23]. We explored the secondary inclusion phenotype of IncA-positive recombinant strains; this

analysis was not possible in strains that are IncA-negative, because our readout of secondary inclusions is dependent on antibodies to IncA. Of the eight IncA-positive recombinant strains tested, recombinants RC-J/953 and RC-L2/971 showed extensive secondary inclusion production (Table 1, Figure 7). These results are surprising because both parental strains (J/6276 and L2-434) used to create RC-J/953 and 3-oxoacyl-(acyl-carrier-protein) reductase RC-L2/971 are low secondary inclusion formers [23]. Recombinant progeny with high secondary inclusion phenotypes where both parents exhibit low secondary inclusion formation suggest a possible interaction

between at least two chlamydial proteins, or at least two independent genetic markers, in the manifestation of the secondary inclusion phenotype. Figure 7 Fluorescent microscopic analysis of the secondary inclusion formation phenotype of recombinant strain RC-J/953. McCoy cells were infected at an MOI of ~0.5, and images were taken 48 h post-infection. All cells were labeled with anti-IncA (green), and anti-OmpA (red), and DNA is labeled with DAPI (blue). A representative secondary inclusion is indicated by the white arrow in the bottom panel. The strain being analyzed is shown at the right of each image. Scale bar, 10 μm. Quantitative analysis of possible loci associated with the secondary inclusion phenotype was inconclusive. This was a function of both the low number of recombinants available for analysis, and the fact the apparently multiple alleles are involved.