The development of axenic culture conditions has considerably facilitated the study of pathogenic mycoplasmas under laboratory conditions. Yet limited information is available regarding the factors that are involved in their virulence and in their interaction with the host, mainly because of the lack of cellular or small-animal models. In an attempt to fill this gap, a model system using the HeLa human epithelial cell line was developed to study the basic interactions between M. agalactiae and eukaryotic cells. We further used this model system, combined with the production of a large mutant library, in a high-throughput screening strategy for the identification of M. agalactiae growth-deficient mutants and mapped 18 regions on the M. agalactiae chromosome that are specifically required for optimal proliferation under cell culture conditions but are dispensable for propagation in axenic medium.
The number of mutants tested was 2.4 times the total number of CDSs found in the genome of the PG2 strain (877 kb; 742 CDSs). Whether the mutant library produced in M. agalactiae
may be approaching saturation is unknown; however, the number of mutants collected is in agreement with the experimental estimations of the minimal size of a mutant library to reach saturation mutagenesis of all nonlethal insertion sites in other mycoplasma species (15
). In the human urogenital pathogen M. genitalium
(580 kb; 475 CDSs), the number of unique transposon insertion sites drops dramatically after 600 mutants, corresponding to 1.3 times of the total number of CDSs found in this organism; whereas in the murine pathogen M. pulmonis
(964 kb; 782 CDSs), the number of inactivating insertions in genes larger than 1 kb nearly reached a plateau at around 1,800 insertion sites (2.3 times the total number of CDSs). High-throughput screening of the M. agalactiae
library using the cell system developed here identified a series of 23 mutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in the presence of HeLa cells. The efficiency of this screening was confirmed by (i) the identification of several growth-deficient mutants sharing identical insertion sites, such as mutants T05.084 and T05.094 that were present at low frequency (<1.4%) in the mutant population originating from one transformation event, and (ii) the identification of growth-deficient mutants having a transposon inserted in the same CDS but at different positions.
The availability of the annotated genome sequence of M. agalactiae
allowed rapid mapping of the transposon insertion sites of selected mutants. Disrupted CDSs for which a predicted function was assigned correspond to 40% of the total number of CDSs and belong to a broad number of functional categories, often with no obvious correlation between the predicted function and its potential role in M. agalactiae
survival under cell culture conditions. Yet several related functions, such as protein folding, iron-sulfur cluster biosynthesis, and DNA repair, have been associated with virulence or stress tolerance in a number of pathogenic bacteria (1
). Another 40% of CDSs encode hypothetical products, many of which were shown to display membrane lipoprotein features and/or to have undergone horizontal gene transfer (HGT) with the mycoides cluster (Fig. ). Two of these proteins belong to a gene family, the drp
, which encodes related proteins containing one or several DUF258 domains. This domain is of unknown function and is found in some bacteria but not in mollicutes, with the exception of the mycoides cluster (45
). In a recent study comparing M. agalactiae
strains using whole-genomic and proteomic approaches, the differential expression of some drp
genes was found in association with the membrane fraction. These data suggested that this family may participate in generating surface diversity, with some drp
genes presenting features of lipoproteins and being expressed and others serving as sequence reservoirs (34
). Interestingly, the drp
genes are part of the gene pool which has undergone HGT with members of the phylogenetically distant mycoides cluster. This cluster contains only ruminant-pathogenic mycoplasma species, and in the absence of a cell wall, surface-exposed lipoproteins may play an important role in mediating interactions with the host. Further studies are needed to confirm the role of these CDSs in the M. agalactiae
interaction with mammalian cells, but they provide an interesting subset of mutants that can reasonably be analyzed in vivo
. Remarkably, a number of integration events occurred in pseudogenes or in NCR that map within a particular 20-kb locus containing a vestige of an integrative conjugative element, ICEA. The implication of an ICEA-related open reading frame (ORF) in the M. agalactiae
interaction with HeLa cells remains to be confirmed. However, it is worth noting that best alignments for ICEA products of the PG2 strain were consistently obtained with the ICEC counterparts of M. capricolum
), a member of the mycoides cluster which causes similar symptoms. Defining whether the growth deficiency phenotype observed in selected mutants resulted from the single transposon insertion or from phase variation or spontaneous mutation of other unrelated genes is essential, especially if regions apparently deprived of CDSs are involved. However, this is hampered by the difficulties in the genetic manipulation of these organisms. These difficulties have been overcome so far by complementation with the two mutants that had the most extreme phenotype.
Complementation studies confirmed that key functions conditioning M. agalactiae
survival and proliferation under cell culture conditions were encoded by the NIF locus. In M. agalactiae
, the locus is composed of two CDSs encoding homologues of nitrogen fixation proteins, NifS and NifU, two proteins involved in iron-sulfur [Fe-S] cluster biosynthesis. Documented in various organisms (14
), [Fe-S] cluster assembly systems are poorly understood in Gram-positive bacteria. Recent studies with Enterococcus faecalis
identified the SUF machinery as the only [Fe-S] cluster biosynthetic system present in the Firmicutes
). As expected by the taxonomic position of mycoplasmas, sequence features of the SUF machinery were identified in M. agalactiae
NifS and NifU proteins. These include the amino acid sequence RSGIFCA surrounding Cys343 of MAG0720 (NifS), which is indicative of a group II (SUF-type) bacterial cysteine desulfurase, whose consensus sequence is RXGHHCA; this sequence clearly distinguishes NifS from group I enzymes of the iron-sulfur cluster, ISC (IscS-type), that display the sequence signature SSGSAC(T/S)S. Similarly, the MAG0730 (NifU) product and SufU scaffold proteins share several features that distinguish them from IscU homologues. They both lack the LPPVK motif present in IscU and contain an 18- to 21-amino-acid insertion between the second and the third conserved cysteine residues. However, despite important sequence homologies with bacterial SUF machineries, mycoplasma NIF proteins exhibit several unique features.
The NIF locus present in mycoplasmas is a simplified version of more complex SUF operons and may encode cysteine desulfurases and scaffold proteins with unique biochemical properties. Its strict conservation among all mycoplasma genomes sequenced so far emphasizes its biological importance in mollicutes. Recent studies with a number of pathogenic bacteria, including Mycobacterium tuberculosis
, Shigella flexneri
, and the plant pathogen Erwinia chrysanthemi
, have established a link between [Fe-S] cluster biosynthesis and virulence (24
). The central role played by bacterial SUF machineries in resistance to iron limitation and oxidative stress suggest that the NIF locus might play a similar role in M. agalactiae
-host interactions. However, preliminary in vitro
studies with M. agalactiae
failed to reveal a particular susceptibility of NIF mutants when bacteria were exposed to oxidative stress or iron limitation. In vivo
studies are in progress to determine the virulence of the NIF mutants in the animal host. The potential implication of this locus in a broad number of processes involving [Fe-S] proteins considerably increases the functions that can be affected in the NIF mutants. The additional functions that have been attributed to cysteine desulfurases (30
), such as the biosynthesis of selenoproteins and multiple cofactors (biotin, lipoic acid, molybdopterin, thiamine, and NAD) as well as iron homeostasis and tRNA modifications, make the situation even more complex.
The growth-deficient phenotype exhibited by the NIF mutants under cell culture conditions and also potentially by other mutants (Fig. ) revealed the decisive role played by metabolic functions in the adaptation of M. agalactiae
to changing environments. A link between carbon metabolism and pathogenicity in mycoplasmas has already been suggested by several groups (5
). The availability of a carbon source in vivo
and its influence on bacterial pathogenicity has reignited interest in using carbon metabolic pathways as viable targets for antibiotic development (7
). This might be particularly important for mycoplasmas, which have limited metabolic capacities and are dependent on the host for many nutrients (37
Attempts to define the minimal amount of genetic information in various mycoplasma species revealed several discrepancies since orthologous genes found to be essential in one organism may be dispensable in another. This is illustrated here by the NIF locus, which was essential for axenic growth of M. genitalium
) but apparently dispensable in other mycoplasma species such as M. pneumoniae
, M. pulmonis
, and M. agalactiae
). A number of situations may account for the occurrence of transposition events in essential genes. The identification of a paralog in M. pulmonis
of the cysteine desulfurase-encoding gene provided a simple explanation for the apparent dispensability of the NIF locus in this species (15
). Given the central role played by [Fe-S] proteins in a variety of fundamental biological processes, the absence of a paralog in the M. agalactiae
genome suggests that essential functions might be performed by unrelated or very distantly related nonorthologous proteins.
The understanding of the basic molecular mechanisms underlying cellular life is of broad interest, and considerable effort has been devoted to establishing candidate minimal genomes. Our study provides a means for addressing this issue at a higher level of complexity, the host-cell context, and new opportunities to decipher mycoplasma-host interaction and virulence.