Stenotrophomonas maltophilia isolata da pazienti affetti da fibrosi cistica : analisi genotipica e caratterizzazione molecolare di determinanti di virulenza
Stenotrophomonas maltophilia is a an environmental organism, that in
recent years has been isolated with increasing frequency as an opportunistic
pathogen in nosocomial infections as well as from the airways of cystic
fibrosis (CF) patients. Despite its growing clinical importance, the
mechanisms adopted by this organism to establish persistent infections have
not been clarified yet. The availability of information on genome sequence
of two S. maltophilia strains (K279a and R551-3), allow development of
new post-genomic approaches direct to understanding of key factors
involved in the colonization of respiratory tract of CF patients and of
cellular and molecular mechanisms of its pathogenicity. Identification of S.
maltophilia genes expressed inside host, is essential for development of new
antimicrobial compounds. Moreover, molecular and genetic characterization
of S. maltophilia clinical strains is essential to understand pathogenicity
mechanism of these organisms and to build detailed epidemiological maps
to draw their spread in CF patients.
The present study is aimed at determining the genomic and
epidemiological relatedness of 52 S. maltophilia strains. Strains included 43
clinical collected, between January 2003 and December 2005, from 43
independent patients (41 were from CF patients, and two from blood
cultures of two non-CF patients) attending the Paediatric Hospital
“Bambino Gesù” of Rome. Other strains were: K279a and LMG958, two S.
maltophilia reference clinical strains, and seven strains of environmental
origin (two isolated within the Hospital “Bambino Gesù”). In these strains
the presence and expression of the virulence-associated genes were
evaluated and their contribution to virulence was assessed using larvae of
the wax moth Galleria mellonella as an infection model. To confirm a role
of the putative virulence factors identified we constructed K279a (the
clinical reference strain whose genome sequence is available) mutants and
performed complementation assay with the wild type functional gene.
Moreover, to study genomic evolution of S. maltophilia toward a
pathogenic lifestyle, we compare the genome of an environmental strain
with that of the clinical strain K279a.
Genotyping analysis. Data that we obtained indicates PFGE
fingerprinting as the most discriminatory technique to characterize
genomospecies present in S. maltophilia. All strains produced well-defined
PFGE profiles: forty-seven different PFGE pulsotypes were identified,
indicating a remarkable genomic diversity among the strains analysed.
Three clusters (pulsotype 1, 17 and 33) showed a banding pattern identical
to at least 1 other isolate likely indicating either cross-transmission among
patients or infection from a common source. However, the results obtained
with the RFLP of gyrB gene and with the variable regions of the 16S rRNA
gene clearly indicated that certain phylogenetic groups are likely better able
to cause infection than others.
Identification and characterization of virulence genes involved in S.
maltophilia pathogenicity. All 52 S. maltophilia strains were characterized
for the presence and expression of type-1 fimbria, esterase, proteases
(StmPr1 and StmPr2). Regarding the type-1 fimbria, only the clinical strains
showed the presence of the gene coding for this surface structure, indicating
that it might be an important virulence factor of this opportunistic pathogen.
The expression of esterase activity by most CF-derived S. maltophilia
strains, reinforces the hypothesis that esterase, such as fimbria, may play
some role in the virulence. In order to verify the role of these potential
virulence factors in S. maltophilia pathogenesis, representative S.
maltophilia strains unable to express proteases or esterase were assayed in
G. mellonella infection. The results indicate that proteases may be relevant
virulence factors of S. maltophilia. Moreover, the type-1 fimbria could play
an important role since the environmental strains, lacking of smf-1, show
the higher lethal dose (LD50).
To analyze the relative role of the two proteases (StmPr1 and StmPr2),
the OBGTC23 strain, naturally impaired in protease expression, was
complemented with the StmPr1 or StmPr2 functional genes and tested in G.
mellonella assay. Data obtained showed that StmPr1 protease play a major
role in S. maltophilia pathogenesis.
Analysis of the role of S. maltophilia virulence factors by mutants
construction. In the complex, the results that we obtained using different
clinical OBGTC strains, impaired in the expression of one or more
hypothetical virulence factors, indicates proteases as important virulence
determinants for S. maltophilia. Nevertheless, these natural mutant strains
could lack of many others virulence determinants that may be involved in
pathogenesis process. Then, to study the effect of the single factors
identified and to confirm their role, deletions encompassing different
putative virulence genes are introduced by allelic exchange in reference
strain K279a, whose genome sequence is available. The proteases mutants
showed a strong reduction of the protease activity and were less virulent,
compared to wild-type, in G. mellonella larvae, confirming that proteases,
particularly StmPr1, are important virulence factors for S. maltophilia. The
proteases expression is controlled by the diffusible signal factor (DSF) a
quorum sensing signal molecule that controls the expression of several
virulence factors. Moreover, the DSF mutant reveals a greater reduction of
virulence in G. mellonella infection model, compared to that of proteases
mutants, suggesting a DSF involvement in the control of proteases but also
of other virulence factors. Finally, despite fimbriae are important to
establish chronic infection in airways CF patients, this surface structures
may be not important in G. mellonella infection model.
Genomic evolution of S. maltophilia toward a pathogenic lifestyle. To
identify the specific genetic determinants acquired from S. maltophilia
during its evolution towards a more pathogenic lifestyle, we performed a
Suppression Subtractive Hybridization: this technique allowed us to identify
the genetic sequences present in the clinical strain K279a and absent in the
environmental strain LMG959. The majority of the subtracted sequence
may have been acquired from other organisms by horizontal gene transfers,
since their different G+C content and since the presence of several IS
elements. Among the sequence, particularly interesting are genes coding for
haemagglutinin, Clp protease, TonB dependent receptor protein, and a
putative ankyrin repeat-containing protein, which are known to be important
virulence factors in many gram-negative bacteria. Moreover, many of the
subtracted sequence represent genes involved in metabolism, DNA
restriction/modification system, transmembrane proteins, hypothetical
proteins and proteins with unknown function. These differences between S.
maltophilia K279a and S. maltophilia LMG959 could be related to niche
adaptation or host preference and this accessory genome may represents an
advantage for pathogen evolution driven by the need for continuous
adaptation to the host in order to evade or suppress coevolving host defense
mechanisms, making it an emergent opportunistic pathogen in nosocomial
infections