Association between transcript levels of the Pseudomonas aeruginosa regA, regB, and toxA genes in sputa of cystic fibrosis patients

Abstract

In this study, we examined the regulation of exotoxin A (ETA) production by Pseudomonas aeruginosa during chronic lung infections of cystic fibrosis (CF) patients. We used a recently developed technique termed population transcript accumulation in hybridization studies with RNA extracted from sputa. With this technique, we demonstrated that the structural gene for ETA, toxA, as well as two genes encoding positive regulators of ETA synthesis, regA and regB, were expressed in the lungs of CF patients infected with P. aeruginosa. These genes were always expressed together, never alone or in pairs, suggesting coincident expression and a possible regulatory role for regA and regB in this environment. Fluctuations in the levels of the three gene products were observed among samples, consistent with a regulatory phenomenon. The level of regB RNA detected never exceeded that ofregA, although the ratio of regA RNA to regB RNA detected did change between samples. These observations are in agreement with in vitro observations which have shown that regB is located 3' to regA in an operon which is expressed from two independently regulated promoters located upstream of regA. The presence of high levels of toxA, regA, and regB RNAs in some sputum samples prompted us to look for hyperproducing-toxin strains in the sputa of CF patients. In vitro, one such strain, 4384, had a transcript accumulation pattern for toxA, regA, and regB similar to that of a laboratory hyperproducer of ETA, strain PA103. These observations suggest that regA and regB are involved in the regulation of ETA production in strains of P. aeruginosa infecting the lungs of CF patients and that some of these strains may regulate ETA production in a manner similar to that of the hyperproducing-ETA strain PA103.

Pseudomonas aeruginosa lasR Transcription Correlates with the Transcription of lasA , lasB , and toxA in Chronic Lung Infections Associated with Cystic Fibrosis

“…Thus, one might expect that we would have difficulties finding correlations between expression of genes even if they were regulated by the same transcriptional activator. However, in the current study and in past studies (38,45) we have found statistically significant correlations between the transcript accumulations for various genes. These correlations occurred even with large sample sizes from a diverse set of patients (Table 1) (45).…”

“…The correlation of lasR population transcript accumulation to toxA transcript accumulation (Table 1) is also curious in light of the previous finding that there is a strong association between expression of the regAB operon and toxA in the lungs of patients with CF (38). In P. aeruginosa PAO1, LasR has been shown to enhance toxA transcription (48).…”

“…These densities of bacteria should produce concentrations of autoinducer that would trigger the expression of the target genes. Furthermore, three genes that are regulated by the LasR-LasI-PAI-1 regulatory system, lasA, toxA, and lasB (13,14,33), are transcribed (38,45,46) and expressed (15,17,19,21,28) in the lungs of patients with CF. Thus, two questions arose which we wanted to address in this study.…”

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa

“…Production of ETA is regulated in vitro (Liu, 1973;Bjorn et al, 1979;Blumenthals et al, 1987) and probably in vivo as well (Raivio et al, 1994). In the laboratory, maximal production occurs in stationary phase when the bacteria are grown at 328C under low-iron conditions (Liu, 1973).…”

Expression of SA5K, a secretion antigen ofMycobacterium tuberculosis, inside human macrophages and in sputum from tuberculosis patients

“…In contrast, very little is yet known about the genes and the corresponding protein products required by M. tuberculosis for growth in the human host [10]. Detection of gene transcripts directly in sputum samples, has successfully been used for the analysis of virulence genes expressed by Pseudomonas aeruginosa or by Staphylococcus aureus in the course of pulmonary infection of patients with cystic fibrosis [11–13]. Recently, analysis of genes expressed in sputum samples from patients with TB has also been performed and it has been suggested as a suitable marker to detect viable M. tuberculosis for rapid diagnosis of pulmonary TB [14,15].…”