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.
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.
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
.
Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. ...Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of
harbors two T6SS gene clusters ...(T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in
. Single deletions of the genes encoding the HU subunits (
and
) decreased mRNA levels of both T6SS. In contrast, the
double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the
genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were
-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria.
T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche.
expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in
, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on
pathogenesis.
serotype Typhimurium is a bacterium that causes gastroenteritis and diarrhea in humans. The genome of
Typhimurium codes for diverse virulence factors, among which are the toxin-antitoxin (TA) ...systems. SehAB is a type II TA, where SehA is the toxin and SehB is the antitoxin. It was previously reported that the absence of the SehB antitoxin affects the growth of
Typhimurium. In addition, the SehB antitoxin can interact directly with the SehA toxin neutralizing its toxic effect as well as repressing its own expression. We identified conserved residues on SehB homologous proteins. Point mutations were introduced at both N- and C-terminal of SehB antitoxin to analyze the effect of these changes on its transcription repressor function, on its ability to form homodimers and on the virulence of
Typhimurium. All changes in amino acid residues at both the N- and C-terminal affected the repressor function of SehB antitoxin and they were required for DNA-binding activity. Mutations in the amino acid residues at the N-terminal showed a lower capacity for homodimer formation of the SehB protein. However, none of the SehB point mutants were affected in the interaction with the SehA toxin. In terms of virulence, the eight single-amino acid mutations were attenuated for virulence in the mouse model. In agreement with our results, the eight amino acid residues of SehB antitoxin were required for its repressor activity, affecting both homodimerization and DNA-binding activity, supporting the notion that both activities of SehB antitoxin are required to confer virulence to
.
The flagella of enteropathogenic
(EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella ...expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (
of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco's Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced
transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of
transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate
expression in order to favor motility and host colonization.
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