Structure-Function Analysis of the Periplasmic Escherichia coli Cyclophilin PpiA in Relation to Biofilm Formation

2017 ◽  
Vol 27 (4) ◽  
pp. 228-236 ◽  
Author(s):  
Aggeliki Skagia ◽  
Eleni Vezyri ◽  
Konstantinos Grados ◽  
Anastasia Venieraki ◽  
Michael Karpusas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Wild Type Strain ◽  
Function Analysis ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Ppiase Activity ◽  
Biofilm Growth ◽  
Negative Effect ◽  
Domains Of Life

The presence of peptidyl-prolyl <i>cis</i>/<i>trans</i> isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions<b> </b>are still not clearly defined. Here, we demonstrate that the &#x0394;<i>ppiA</i> deletion strain from <i>Escherichia coli</i> exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the &#x0394;<i>ppiA</i> strain during biofilm growth.

scholarly journals Synthesis in Escherichia coli of two smaller enzymically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene

1998 ◽  
Vol 335 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Yin-yuan MO ◽  
J. SESHU ◽  
Dong WANG ◽  
Louis P. MALLAVIA
Keyword(s):  
Escherichia Coli ◽  
Coxiella Burnetii ◽  
Sequence Homology ◽  
Degradation Products ◽  
Open Reading Frame ◽  
Site Directed Mutagenesis ◽  
Ppiase Activity ◽  
Reading Frame ◽  
Macrophage Infectivity Potentiator ◽  
Macrophage Infectivity

FK506-binding proteins (FKBPs) have been identified in a variety of eukaryotic and prokaryotic organisms. Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Coxiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (PPIase) activity. Further characterization of the cbmip gene has identified two additional proteins with molecular masses of 15.5 and 15.0 kDa that are synthesized, in addition to the 23.5 kDa CbMip, when expressed in Escherichia coli. Amino acid sequencing at the N-terminus combined with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation. When the internal methionines serving as start sites were replaced with lysine by site-directed mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was abolished even though the synthesis of 23.5 kDa CbMip was intact. This confirmed that the 15.5 and 15.0 kDa proteins are indeed generated by translational reinitiation and are not degradation products of the 23.5 kDa protein. Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit PPIase activity. Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15.0 kDa proteins are designated as C. burnetiiFKBP (Cb-FKBP) analogues I and II, respectively. TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indicating that structural variations could allow for different subcellular compartmentalization of similar proteins. Western-blot analysis of lysates of purified C. burnetii using a CbMip-specific monoclonal antibody revealed the presence of a protein migrating at ≈ 15 kDa, indicating the presence of smaller Cb-FKBP analogue(s) in C. burnetii, although at much lower levels compared with 23.5 kDa CbMip. This unique gene organization seen with cbmip may provide the organism with a mechanism of efficient use of its limited genetic information to synthesize proteins that are structurally different yet functionally similar.

scholarly journals Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

2019 ◽  
Author(s):  
Philippe Vogeleer ◽  
Antony T. Vincent ◽  
Samuel M. Chekabab ◽  
Steve J. Charette ◽  
Alexey Novikov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Inorganic Phosphate ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Wild Type ◽  
E Coli ◽  
Pi Starvation ◽  
Pst System

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. InE. coliO157:H7, the Δpstmutant, biofilm, and autoagglutination were increased. In the double-deletion mutant ΔpstΔphoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from Δpstmutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation andpstmutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the Δpstmutant. Interestingly,waaH, encoding a glycosyltransferase associated with LPS modifications inE. coliK-12, was highly expressed in the Δpstmutant ofE. coliO157:H7. Deletion ofwaaHfrom the Δpstmutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the Δpstmutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how theE. coliO157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

scholarly journals Whole slide imaging is a high-throughput method to assess Candida biofilm formation

2020 ◽  
Author(s):  
Maximilian W. D. Raas ◽  
Thiago P. Silva ◽  
Jhamine C. O. Freitas ◽  
Lara M. Campos ◽  
Rodrigo L. Fabri ◽  
...  
Keyword(s):  
Single Cell ◽  
Biofilm Formation ◽  
Large Scale ◽  
Structural Features ◽  
Whole Slide Imaging ◽  
Bright Field ◽  
Biofilm Growth ◽  
Fluorescent Markers ◽  
Golden Standard ◽  
Important Addition

AbstractNew strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyze fungal biofilm growth.

scholarly journals RstA, a two-component response regulator, plays important roles in multiple virulence-associated processes in enterohemorrhagic Escherichia coli O157:H7

Gut Pathogens ◽  
2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Yutao Liu ◽  
Shujie Li ◽  
Wendi Li ◽  
Peisheng Wang ◽  
Peng Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acid Tolerance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Regulatory Function ◽  
Wild Type ◽  
Two Component

Abstract Background Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157. Results In the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP. Conclusions In summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections.

scholarly journals Mat fimbriae promote biofilm formation by meningitis-associated Escherichia coli

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2408-2417 ◽  
Author(s):  
Timo A. Lehti ◽  
Philippe Bauchart ◽  
Johanna Heikkinen ◽  
Jörg Hacker ◽  
Timo K. Korhonen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Early Stage ◽  
Surface Expression ◽  
Growth Media ◽  
Biofilm Growth ◽  
Abiotic Surfaces ◽  
K 12 ◽  
Biofilm Development

The mat (or ecp) fimbrial operon is ubiquitous and conserved in Escherichia coli, but its functions remain poorly described. In routine growth media newborn meningitis isolates of E. coli express the meningitis-associated and temperature-regulated (Mat) fimbria, also termed E. coli common pilus (ECP), at 20 °C, and here we show that the six-gene (matABCDEF)-encoded Mat fimbria is needed for temperature-dependent biofilm formation on abiotic surfaces. The matBCDEF deletion mutant of meningitis E. coli IHE 3034 was defective in an early stage of biofilm development and consequently unable to establish a detectable biofilm, contrasting with IHE 3034 derivatives deleted for flagella, type 1 fimbriae or S-fimbriae, which retained the wild-type biofilm phenotype. Furthermore, induced production of Mat fimbriae from expression plasmids enabled biofilm-deficient E. coli K-12 cells to form biofilm at 20 °C. No biofilm was detected with IHE 3034 or MG1655 strains grown at 37 °C. The surface expression of Mat fimbriae and the frequency of Mat-positive cells in the IHE 3034 population from 20 °C were high and remained unaltered during the transition from planktonic to biofilm growth and within the matured biofilm community. Considering the prevalence of the highly conserved mat locus in E. coli genomes, we hypothesize that Mat fimbria-mediated biofilm formation is an ancestral characteristic of E. coli.

scholarly journals The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1316-1331 ◽  
Author(s):  
Christophe Beloin ◽  
Kai Michaelis ◽  
Karin Lindner ◽  
Paolo Landini ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Mg1655 ◽  
Upec Strain ◽  
E Coli ◽  
Initial Adhesion ◽  
State Production ◽  
K 12

ABSTRACT We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5 Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

scholarly journals Identification of Type 3 Fimbriae in Uropathogenic Escherichia coli Reveals a Role in Biofilm Formation

2007 ◽  
Vol 190 (3) ◽  
pp. 1054-1063 ◽  
Author(s):  
Cheryl-Lynn Y. Ong ◽  
Glen C. Ulett ◽  
Amanda N. Mabbett ◽  
Scott A. Beatson ◽  
Richard I. Webb ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Klebsiella Oxytoca ◽  
The United States ◽  
Conjugative Plasmid ◽  
Biofilm Growth ◽  
Indwelling Catheters ◽  
E Coli ◽  
Type 3

ABSTRACT Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens.

scholarly journals Global Gene Expression Profiling of Asymptomatic Bacteriuria Escherichia coli during Biofilm Growth in Human Urine

2006 ◽  
Vol 75 (2) ◽  
pp. 966-976 ◽  
Author(s):  
Viktoria Hancock ◽  
Per Klemm
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Expression Profile ◽  
Human Urine ◽  
Biofilm Formation ◽  
Expression Profiles ◽  
Asymptomatic Bacteriuria ◽  
Knockout Mutant ◽  
Biofilm Growth ◽  
E Coli

ABSTRACT Urinary tract infection (UTI) is an important health problem worldwide, with many millions of cases each year, and Escherichia coli is the most common organism causing UTI in humans. Also, E. coli is responsible for most infections in patients with chronic indwelling bladder catheter. The two asymptomatic bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm formers in their natural growth medium, human urine, than the two uropathogenic E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the expression profile during biofilm growth in urine of the two ABU strains 83972 and VR50. Significant differences in expression levels were seen between the biofilm expression profiles of the two strains with the corresponding planktonic expression profiles in morpholinepropanesulfonic acid minimal laboratory medium and human urine; 417 and 355 genes were up- and down-regulated, respectively, during biofilm growth in urine of 83972 and VR50. Many genes involved in transcription and stress were up-regulated in biofilm-grown cells. The role in biofilm formation of four of the up-regulated genes, i.e., yceP, yqgA, ygiD, and aaeX, was investigated by creating single-knockout mutant versions of 83972 and VR50; all mutants showed reduced biofilm formation in urine by 18 to 43% compared with the wild type (P < 0.05). Also, the expression profile of strain 83972 in the human urinary tract partially overlaps with the biofilm expression profile.

Cellular chain formation in Escherichia coli biofilms

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1407-1417 ◽  
Author(s):  
Rebecca Munk Vejborg ◽  
Per Klemm
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Immunofluorescence Microscopy ◽  
Asymptomatic Bacteriuria ◽  
Type I ◽  
Chain Formation ◽  
Biofilm Growth ◽  
E Coli ◽  
Type I Fimbriae ◽  
K 12

In this study we report on a novel structural phenotype in Escherichia coli biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular interactions facilitated by antigen 43 (Ag43), a self-associating autotransporter (SAAT) protein, which has previously been implicated in auto-aggregation and biofilm formation. Immunofluorescence microscopy suggested that Ag43 was concentrated at or near the cell poles, although when the antigen was highly overexpressed, a much more uniform distribution was seen. Immunofluorescence microscopy also indicated that other parameters, including dimensional constraints (flow, growth alongside a surface), may also affect the final biofilm architecture. Moreover, chain formation was affected by other surface structures; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E. coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species.

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