Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection

Abstract

Mycoplasma hominis is the second smallest facultative pathogen of the human urogenital tract. With less than 600 protein-encoding genes, it represents an ideal model organism for the study of host-pathogen interactions. For a comprehensive characterisation of the M. hominis action in infection a customized Mho microarray, which was based on two genome sequences (PG21 and LBD-4), was designed to analyze the dynamics of the mycoplasma transcriptome during infection and validated for M. hominis strain FBG. RNA preparation was evaluated and adapted to ensure the highest recovery of mycoplasmal mRNAs from in vitro HeLa cell infection assays. Following cRNA hybridization, the read-out strategy of the hybridization results was optimized and confirmed by RT-PCR. A statistically robust infection assay with M. hominis strain FBG enabled the identification of differentially regulated key effector molecules such as critical cytoadhesins (4 h post infection (pI)), invasins (48 h pI) and proteins associated with establishing chronic infection of the host (336 h pI). Of the 294 differentially regulated genes (>2-fold) 128 (43.5%) encoded hypothetical proteins, including lipoproteins that seem to play a central role as virulence factors at each stage of infection: P75 as a novel cytoadhesin candidate, which is also differentially upregulated in chronic infection; the MHO_2100 protein, a postulated invasin and the MHO_730-protein, a novel ecto-nuclease and domain of an ABC transporter, the function of which in chronic infection has still to be elucidated. Implementation of the M. hominis microarray strategy led to a comprehensive identification of to date unknown candidates for virulence factors at relevant stages of host cell infection.

Fig 1. Clustering of differentially regulated M. hominis FBG genes at each time point of HeLa cell infection.

A, ratio of cytoadhesive M. hominis cells to HeLa cells (MOI 50) and at each time point of infection (1 h, 4 h, 48 h and 336 h); B, principal component analysis; C, hierarchical clustering of the differentially regulated FBG genes at each time point, measured in biological duplicates.

Fig 2. Distribution of differentially regulated M. hominis FBG genes.

Numbers of differentially up- and downregulated (> 2-fold) genes of M. hominis strain FBG at 4 h, 48 h and 336 h post infection compared to time point 1 h. A, numbers of differentially up- and downregulated genes; B, Venn diagram of differentially upregulated genes; C, Venn diagram of differentially downregulated genes.

Fig 3. Pathway-dependent transcript regulation.

Differentially regulated M. hominis FBG genes at 4 h, 48 h and 336 h pI were sorted to KEGG-pathway maps and integrated into those belonging to the Metabolism, Genetic or Environmental Information Processing and Cellular Processes. The number of differentially up- and downregulated pathway genes >2 fold are shown.

Fig 4. Comparison of microarray and RT-qPCR results.

Total RNA of M. hominis-infected HeLa cells for 4 h, 48 h or 336 h was subjected to Mho-microarray or RT-qPCR analyses and the change in expression levels of the named genes, with respect to that at the start of infection (1 h for RNAs of 08/15 and 0 h for RNAs of 04/11 and 11/11), quantified as described in the Method section.

Fig 5. RT-PCR analysis of the postulated ABC transporter genes 730–760.

The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used (Table 1) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).