Open AccessValidation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection
Author(s) -
Birgit Henrich,
Freya Kretzmer,
René Deenen,
Karl Köhrer
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0181383
Subject(s) - biology , mycoplasma hominis , microarray , transcriptome , effector , pathogen , virulence , microbiology and biotechnology , microarray analysis techniques , gene , virology , gene expression , mycoplasma , genetics , immunology
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.