The genome of
Mycobacterium tuberculosis
(
Mtb
) harbors the genetic machinery for assembly of the
F
imbrial
l
ow-molecular-weight
p
rotein (Flp) type IV pilus. Presumably, the Flp pilus is essential ...for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (
tadZ, tadA, tadB, tadC, flp, tadE
, and
tadF
) in
Mtb
growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of
tad/flp
genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of
Mtb
with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.
In enteropathogenic
(EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to ...investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (Δ
, Δ
, Δ
, Δ
, Δ
, and Δ
), and in secreting effector molecules (Δ
and Δ
) were tested for flagella production under several growth conditions. The Δ
mutant did not produce flagella in any condition tested, although
was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The Δ
and Δ
mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of
transcription. The Δ
, Δ
, Δ
, Δ
, and Δ
mutants produced low levels of flagella, suggesting these regulators are activators of
expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.
The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence ...have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host’s ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium.
Summary
Enterotoxigenic Escherichia coli produces a long type 4 pilus called Longus. The regulatory elements and the environmental signals controlling the expression of Longus‐encoding genes are ...unknown. We identified two genes lngR and lngS in the Longus operon, whose predicted products share homology with transcriptional regulators. Isogenic lngR and lngS mutants were considerably affected in transcription of lngA pilin gene. The expression of lngA, lngR and lngS genes was optimally expressed at 37°C at pH 7.5. The presence of glucose and sodium chloride had a positive effect on Longus expression. The presence of divalent ions, particularly calcium, appears to be an important stimulus for Longus production. In addition, we studied H‐NS, CpxR and CRP global regulators, on Longus expression. The response regulator CpxR appears to function as a positive regulator of lng genes as the cpxR mutant showed reduced levels of lngRSA expression. In contrast, H‐NS and CRP function as negative regulators since expression of lngA was up‐regulated in isogenic hns and crp mutants. H‐NS and CRP were required for salt‐ and glucose‐mediated regulation of Longus. Our data suggest the existence of a complex regulatory network controlling Longus expression, involving both local and global regulators in response to different environmental signals.
In the last ten years, Stenotrophomonas maltophilia has gained increasing interest as an important agent of infection, which is why it has come to be recognized as a serious cause of nosocomial ...infections related to bloodstream infections, pneumonia, and cancer, mainly in patients with intensive care, and is associated with high mortality rates in immunocompromised patients, with prolonged hospital stays and extensive use of antimicrobials. The importance of this microorganism lies in its low pathogenicity, high multiresistance to various antibiotics, and frequent and persistent isolation in predisposed patients. In addition, few studies have evaluated its epidemiology and clinical relevance. The pathogenesis of biofilms lies mainly in the fact that they can generate persistent chronic infections that are difficult to eradicate. To this extent, it is important to make the characteristics of the biofilm formation behavior of Stenotrophomonas maltophilia known and generate more knowledge about its colonization or infection in humans through this review, which discusses more recent information.
Enterobacter cloacae
has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several
E. cloacae
strains revealed the presence of genes that ...code for expression of at least one type VI secretion system (T6SS). Here, we report that
E. cloacae
strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes
clpV1
and
hcp1
significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of
E. cloacae
cultures and a functional T6SS-1 was required for the secretion of this protein. A
clpV2
mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent.
In vivo
data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in
clpV1
and
clpV2
genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of
E. cloacae
with specialized functions in the interaction with other bacteria and with host cells.
is a resident of the human gut. However, certain
toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes ...antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the
and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of
, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δ
isogenic mutant. In summary, we found that CRP directly activates the transcription of the
and NRPS operons and that the absence of CRP reduced cytotoxicity of
on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.
In
the expression of type 1 pili (T1P) is determined by the site-specific inversion of the
ON-OFF switch located immediately upstream of major fimbrial subunit gene
. Here we investigated the role of ...virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the
switch in the human enteropathogenic
(EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the
switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆
mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the
,
, and
promoters using
transcriptional fusions indicated that only P
activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the
switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of
.
Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we ...investigated the contribution of curli and cellulose to the adhesive properties of enterohaemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Overexpression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared with growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Δfis produced abundant amounts of curli whereas a double fis/csgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favour host colonization, biofilm formation and survival in different environments.
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