Generation of isogenic K54 capsule‐deficient <i>Escherichia coli</i> strains through Tn<i>pho</i>A‐mediated gene disruption

Russo, Thomas A.; Guenther, Jane E.; Wenderoth, Suzanne; Frank, Michael M.

doi:10.1111/j.1365-2958.1993.tb01696.x

Cited by 60 publications

(87 citation statements)

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“…The model pathogen CP9 is an E. coli blood isolate cultured from a patient with sepsis and has been previously described in detail (19,39). CP9 possesses many of the characteristics of typical ExPEC strains (40) and is highly virulent in a urinary tract infection model (36), an intraperitoneal infection model (42), and a pneumonia model (35,43).…”

Section: Methodsmentioning

confidence: 99%

E. colivirulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model

Russo¹,

Davidson²,

Genagon³

et al. 2005

American Journal of Physiology-Lung Cellular and Molecular Phys

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In this study a wild-type extraintestinal pathogenic strain of E. coli (ExPEC)(CP9) and isogenic derivatives deficient in hemolysin (Hly) and cytotoxic necrotizing factor (CNF) were assessed in vitro and in a rat model of gram-negative pneumonia to test the hypothesis that these virulence factors induce neutrophil apoptosis and/or necrosis/lysis. As ascertained by in vitro caspase-3/7 and LDH activities and neutrophil morphology, Hly mediated neutrophil apoptosis at lower E. coli titers (1 ϫ 10 5-6 cfu) and necrosis/lysis at higher titers (Ն1 ϫ 10 7 cfu). Data suggest that CNF promotes apoptosis but not necrosis or lysis. We also demonstrate that annexin V/7-amino-actinomycin D staining was an unreliable assessment of apoptosis using live E. coli. The use of caspase-3/7 and LDH activities and neutrophil morphology supported the notion that necrosis, not apoptosis, was the primary mechanism by which neutrophils were affected in our in vivo gram-negative pneumonia model using live E. coli. In addition, in vivo studies demonstrated that Hly mediates lung injury. Neutrophil necrosis was not observed when animals were challenged with purified lipopolysaccharide, demonstrating the importance of using live bacteria. These findings establish that Hly contributes to ExPEC virulence by mediating neutrophil toxicity, with necrosis/ lysis being the dominant effect of Hly on neutrophils in vivo and by lung injury. Whether Hly-mediated lung injury is due to neutrophil necrosis, a direct effect of Hly, or both is unclear.Escherichia coli; gram-negative bacilli; cytotoxic necrotizing factor-1 NOSOCOMIAL PNEUMONIA ACCOUNTS for 15% of hospital-acquired infections (2). Gram-negative enteric bacilli such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterobacter sp., Acinetobacter, and Stenotrophomonas are the most common cause of hospital-acquired pneumonia, being implicated in 55-85% of cases (8 -10, 24). Severe disease usually occurs when these agents cause pneumonia, with associated crude mortality and estimated attributable mortality ranging from 24 to 76% and 20 to 50%, respectively, which translates into 36,000 -80,000 deaths annually (2, 8, 24, 52). Over the last 10 -15 years, there has been little improvement in the outcome from this infection. The estimated annual cost of nosocomial pneumonias alone due to gram-negative bacilli in this country is greater than one billion dollars (55).A goal of our laboratory is to identify strategies to decrease the morbidity and mortality caused by gram-negative pneumonia. To accomplish this, we have been studying neutrophil-pathogen interactions in a rat model of bacterial pneumonia, using E. coli as a model pathogen (37,38,43). The importance of neutrophils in protecting against infection in the lung is as great as any other site in the body (46). Increased susceptibility and severity of pneumonia occur in the setting of neutropenia and with genetically inherited (e.g., chronic granulomatous disease) or drug-induced (e.g., steroids) disorders of neutrophil function...

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Section: Methodsmentioning

confidence: 99%

E. colivirulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model

Russo¹,

Davidson²,

Genagon³

et al. 2005

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Primers 406 and 408 (Table 1) designed to this region were subsequently used in PCRs with chromosomal DNA from M. catarrhalis 25238, the previously defined LOS serotype A strain, and M. catarrhalis RS10, the previously defined LOS serotype C strain (Table 2) (3,4). PCR was performed in 50-l reaction mixtures containing PCR SuperMix (Invitrogen, Carlsbad, CA), 20 pmol/l of each primer, and 1 l chromosomal DNA prepared as previously described (26). Amplifications were performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol for 25 cycles with an annealing temperature of 53.1°C and extension time of 4 min.…”

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Multiplex PCR Assay That Identifies the Major Lipooligosaccharide Serotype Expressed by Moraxella catarrhalis Clinical Isolates

Edwards¹,

Schwingel²,

Datta

et al. 2005

J Clin Microbiol

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A heterologous cluster of glycosyltransferase genes was identified in the three Moraxella catarrhalis LOS serotype strains. Multiple PCR primers designed to this region amplified products that differentiate between the serotypes more rapidly and efficiently than previously described serological analyses. This assay will be valuable for clinical and research-based studies.Moraxella catarrhalis, a gram-negative diplococcus, is considered a significant cause of acute otitis media in children and lower respiratory infections in adults with chronic obstructive pulmonary disease (COPD) (12,21,29). A number of putative virulence factors have been described for M. catarrhalis (8,11,12,17,20), including a surface-exposed lipooligosaccharide (LOS) (6,18,23,32). Structural and serological studies with M. catarrhalis have described only three different LOS serotypes (termed A, B, and C), which vary in length and content of the oligosaccharide branches (3-5, 13, 15, 28). One serological study by Vaneechoutte et al. grouped clinical isolates into serotypes A (60%), B (30%), and C (5%), with 5% of the strains unidentified (28). That has been the only study to investigate the prevalence of specific M. catarrhalis LOS serotypes in the population. The difficulties with serological determinations of M. catarrhalis LOS expression are the limited quantities of antibodies, the absolute requirement for purified sample, and the potential for cross-reactivity between serotypes A and C (13,24,25).Recently, a cluster of three glycosyltransferase (lgt) genes were identified and characterized in a strain of M. catarrhalis 7169 expressing serotype B LOS (6). Primers 406 and 408 (Table 1) designed to this region were subsequently used in PCRs with chromosomal DNA from M. catarrhalis 25238, the previously defined LOS serotype A strain, and M. catarrhalis RS10, the previously defined LOS serotype C strain (Table 2) (3, 4). PCR was performed in 50-l reaction mixtures containing PCR SuperMix (Invitrogen, Carlsbad, CA), 20 pmol/l of each primer, and 1 l chromosomal DNA prepared as previously described (26). Amplifications were performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol for 25 cycles with an annealing temperature of 53.1°C and extension time of 4 min. Primers 406 and 408 (Table 1) produced an amplicon of 4.3 kb in serotype A and C strains, which was 1 kb larger than the product amplified in the serotype B strain, 7169 (data not shown).Sequence analyses (MacVector 7.2 software; Accelrys, San Diego, CA) of the entire region amplified by primer 406 and flanking primer 704 (Table 1) identified an additional open reading frame upstream of the original lgt cluster described in 7169 and in the same orientation as lgt1, as illustrated in Fig. 1A and C. A ClustalW alignment with the translated sequence of this open reading frame (Lgt4) in both strains revealed 46% identity and 60% similarity to Lgt1, an ␣(1-2) glucosyltransferase in M. catarrhalis 7169 (6). The 5Ј end of this cluster...

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“…Its effect is greatest at 28°C and results in a functionally significant change in K54 capsule production. (25). Transductions were performed by using the bacteriophage T4 as previously described (25 lon' and Ion-disrupted backgrounds correlated with A6., standardization to the protein concentration was not necessary.…”

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“…(25). Transductions were performed by using the bacteriophage T4 as previously described (25 lon' and Ion-disrupted backgrounds correlated with A6., standardization to the protein concentration was not necessary. Because baseline alkaline phosphatase activity from CP9 was less than 1% of the fusion activity of the strains tested, it was not felt to significantly contribute to the specific activities of the test strains and therefore was not accounted for when Table 2.…”

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An extraintestinal, pathogenic isolate of Escherichia coli (O4/K54/H5) can produce a group 1 capsule which is divergently regulated from its constitutively produced group 2, K54 capsular polysaccharide

Russo

Singh

1993

J Bacteriol

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We are studying an 04/K54/H5 Escherichia coli bacteremic isolate (CP9) as a model pathogen for extraintestinal infection. Its group 2, K54 capsular polysaccharide is an important virulence determinant and confers serum resistance. In this study the effect of the group 1 capsule regulators, RcsA, RcsB, and Lon protease, on the regulation of CP9's capsular polysaccharides was assessed. It was established that in the presence of multicopy rcsA or with disruption of Ion, CP9 can be induced to produce a group 1 capsule. RcsA, RcsB, and Lon are present in this K54 background and regulate group 1 capsule expression in a fashion similar to that described for K-12 strains. Two independent group 2 capsule gene protein fusions (cll.29::TnphoA and c11.137::TnphoA) were used to evaluate the effects of these regulators on group 2 K54 capsule production. Disruption of lon resulted in 1.9-fold (TR293 [cll.29::TnphoA lon-146]) and 3.4-fold (TR1373 [cll.137::TnphoA lon-146]) decreases in fusion activity at 28°C, relative to the baseline level. However, decreases in fusion activity at 42°C were only 1.2-and 1.4-fold, respectively. Inactivation of both Ion and rcsA or Ion and rcsB restored fusion activity to baseline levels at 28°C, but only a partial restoration of activity was seen at higher temperatures. To assess whether these differences in fusion activity reflected a functional change in capsule production, the effects of 80%o normal human serum (NHS) were tested against CP9 and TR93 (ion-146). Since the group 2 K54 capsule protects against the bactericidal activity of 80%Yo NHS, a decrease in its production results in an increase in serum sensitivity. Viable counts of CP9 increased 10-fold in 80%Yo NHS over 3 h at 28°C, as expected. In contrast to CP9, TR93 (ion-146) incurred a 10-fold loss in viability under the same conditions. The levels of RcsA are increased in TR93 (ion-146) as a consequence oflon disruption; therefore, these results in conjunction with the cl1::TnphoA protein fusion data establish RcsA as a negative regulator of the group 2 K54 capsular polysaccharide. Its action appears to be mediated through RcsB and is greatest at 28°C but decreases at higher temperatures. Furthermore, these results also suggest the existence of another Lon-sensitive negative regulator of group 2 K54 capsule production, which is active at higher temperatures.

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Generation of isogenic K54 capsule‐deficient Escherichia coli strains through TnphoA‐mediated gene disruption

Cited by 60 publications

References 31 publications

E. colivirulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model

E. colivirulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model

Multiplex PCR Assay That Identifies the Major Lipooligosaccharide Serotype Expressed by Moraxella catarrhalis Clinical Isolates

An extraintestinal, pathogenic isolate of Escherichia coli (O4/K54/H5) can produce a group 1 capsule which is divergently regulated from its constitutively produced group 2, K54 capsular polysaccharide

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