Carlos Balsalobre

Biofilm infections, their resilience to therapy and innovative treatment strategies

Biofilm formation of microorganisms causes persistent tissue and foreign body infections resistant to treatment with antimicrobial agents. Up to 80% of human bacterial infections are biofilm associated; such infections are most frequently caused by Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus and Enterobacteria such as Escherichia coli. The accurate diagnosis of biofilm infections is often difficult, which prevents the appropriate choice of treatment. As biofilm infections significantly contribute to patient morbidity and substantial healthcare costs, novel strategies to treat these infections are urgently required. Nucleotide second messengers, c‐di‐GMP, (p)ppGpp and potentially c‐di‐AMP, are major regulators of biofilm formation and associated antibiotic tolerance. Consequently, different components of these signalling networks might be appropriate targets for antibiofilm therapy in combination with antibiotic treatment strategies. In addition, cyclic di‐nucleotides are microbial‐associated molecular patterns with an almost universal presence. Their conserved structures sensed by the eukaryotic host have a widespread effect on the immune system. Thus, cyclic di‐nucleotides are also potential immunotherapeutic agents to treat antibiotic‐resistant bacterial infections.

Distribution and characterization of faecal verotoxin-producing Escherichia coli (VTEC) isolated from healthy cattle.

Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo farms were examined for verotoxin-producing Escherichia coli (VTEC). We found VTEC on 95% of the farms. The prevalence rates of VTEC infection in asymptomatic cows and calves were estimated to be 35 and 37%, respectively. The proportion of animals infected on each farm ranged from 0 to 100%. VTEC strains isolated from healthy cattle belonged to 27 O serogroups; however, 57% (85 of 149) were of one of 8 serogroups (O2, O8, O22, O77, O82, O105, O113 and O171). Nearly 60% of the bovine VTEC strains belonged to serogroups that cause haemorrhagic colitis and haemolytic uraemic syndrome in humans. The VTEC were all non-O157:H7; 91% were eae-negative and 86% produced VT2 or VT1 and VT2. These characteristics are different from those of VTEC isolated from calves with diarrhoea.

Release of the type I secreted α-haemolysin via outer membrane vesicles from Escherichia coli

The α‐haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the α‐haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The α‐haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The α‐haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the α‐haemolysin were distinct from the OMVs not containing α‐haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of α‐haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted α‐haemolysin from the bacteria to the host cells during bacterial infections.

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS

Abstract The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP

Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins**

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

Nucleoid Proteins Stimulate Stringently Controlled Bacterial Promoters: A Link between the cAMP-CRP and the (p)ppGpp Regulons in Escherichia coli

We report that the H-NS nucleoid protein plays a positive role in the expression of stringently regulated genes in Escherichia coli. Bacteria lacking both H-NS and the paralog StpA show reduced growth rate. Colonies displaying an increased growth rate were isolated, and mapping of a suppressor mutation revealed a base pair substitution in the spoT gene. The spoT(A404E) mutant showed low ppGpp synthesizing ability. The crp gene, which encodes the global regulator CRP, was subject to negative stringent regulation. The stable RNA/protein ratio in an hns, stpA strain was decreased, whereas it was restored in the suppressor strain. Our findings provide evidence of a direct link between the cAMP-CRP modulon and the stringent response.

(p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB.

In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp‐deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp0 suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp0 strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp‐stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.

Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo

The expression of type 1 fimbriae is dependent on the intracellular levels of ppGpp through stimulation of fimB transcription. Here we show that in contrast to the previously described decreased fimbriation observed in a ppGpp‐deficient strain, DksA deficiency results in a hyperfimbriated state. In vivo assays show that the effect of DksA deficiency on the type 1 fimbriae occurs at the phase variation level because of elevated transcription from the fimB P2 promoter. In contrast, our in vitro transcription studies demonstrate that ppGpp and DksA can stimulate transcription from the fimB P2 promoter both independently and codependently. We provide evidences that the apparently contradictory results from the in vivo and in vitro transcriptional studies are at least in part a consequence of the increased association of the anti‐pausing factors (GreA and GreB) to the RNA polymerase in the absence of DksA in vivo.

The novel Hha/YmoA family of nucleoid‐associated proteins: use of structural mimicry to modulate the activity of the H‐NS family of proteins

The Hha/YmoA family of proteins is a group of conserved, low‐molecular‐weight proteins involved in the regulation of gene expression. Studies performed in Escherichia coli, Salmonella sp. and Yersinia sp. highlight the contribution of these proteins in regulating bacterial virulence, horizontal gene transfer and cell physiology. Genes encoding such proteins are located on chromosomes and plasmids in different genera of Gram‐negative bacteria. Their mode of action is currently being analysed by studying direct binding of Hha to DNA and as a component of protein complexes with regulatory functions. Recent data on the interaction of Hha with the H‐NS family of proteins and structural information suggest a physiological role for such protein complexes in many aspects of gene regulation.