Adenosine has been widely associated with hypoxia of many origins, including those associated with inflammation and tumorogenesis. A number of recent studies have implicated metabolic control of adenosine generation at sites of tissue hypoxia. Here, we examine adenosine receptor control and amplification of signaling through transcriptional regulation of endothelial and epithelial adenosine receptors. Initial studies confirmed previous findings indicating selective induction of human adenosine A2B receptor (A2BR) by hypoxia. Analysis of the cloned human A2BR promoter identified a functional hypoxia-responsive region, including a functional binding site for hypoxia-inducible factor (HIF) within the A2BR promoter. Further studies examining HIF-1alpha DNA binding and HIF-1alpha gain and loss of function confirmed strong dependence of A2BR induction by HIF-1alpha in vitro and in vivo mouse models. Additional studies in endothelia overexpressing full-length A2BR revealed functional phenotypes of increased barrier function and enhanced angiogenesis. Taken together, these results demonstrate transcriptional coordination of A2BR by HIF-1alpha and amplified adenosine signaling during hypoxia. These findings may provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.
It has been shown that the loss of PilD, a prepilin peptidase necessary for type IV pilus biogenesis and establishment of the type II secretion apparatus is associated with loss of virulence in Legionella pneumophila. L. pneumophila is the species most frequently associated with Legionnaires’ disease, but virulence factors unique to this species are not known, so the secretion kinetics of several pilD-dependent enzyme activities, including protease, acid phosphatase, phospholipase A (PLA) and lysophospholipase A (LPLA), of L. pneumophila and non-pneumophila species were compared during growth in BYE broth. Enzyme activity appeared during mid-exponential growth phase and reached maximal levels on entry into stationary growth phase. None of the enzyme activities were unique to L. pneumophila and it did not exclusively secrete the highest amounts of the hydrolytic proteins. However, the timing of PLA and LPLA secretion in L. pneumophila differed compared to other species. PLA activity was secreted prior to LPLA activity in L. pneumophila, which may lead to an accumulation of the cytotoxic agent lysophosphatidylcholine (LPC). In addition to L. pneumophila, several other Legionella species, including Legionella steigerwaltii and Legionella gormanii, were able to enrich for LPC due to a very potent PLA activity accompanied by only moderate LPLA activity. These species, in contrast to L. pneumophila, have not been shown to multiply within monocytic host cells. Thus none of the secreted enzymic activities investigated were unique to L. pneumophila, nor were they secreted at high concentrations. However, the timing of PLA and LPLA secretion may contribute to pathogenicity.
Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.
ABSTRACT Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449–2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii , using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii , significantly enhanced replication of L. gormanii , L. micdadei , L. steigerwaltii , L. longbeachae , and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella -infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella -infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.
The opportunistic pathogen Legionella pneumophila, the etiologic agent of Legionnaires disease, is able to invade and multiply intracellularly in human macrophages. This process is controlled by several bacterial virulence factors. As recently demonstrated, one of these virulence factors, the macrophage infectivity potentiator (Mip) protein, is important for invasion and proper intracellular establishment of L. pneumophila in macrophages and protozoa. Knockout mutants devoid of a functional mip-gene enter host cells much less effectively but intracellular replication is not affected. Using a Pmip-green fluorescent protein reporter construct in L. pneumophila substrain Corby, Pmip was recently shown to be constitutively active in replicating bacteria. A stringent regulation during the infection process could not be observed, neither in intracellular nor in BYE broth-grown bacteria. For enhanced temporal and quantitative resolution, we examined the activity of mip on RNA level in order to detect short transient regulatory events. Our results show that Pmip of L. pneumophila is temporarily repressed directly after invasion of the monocytic human cell line MonoMac 6 and regains activity after 24 h of intracellular replication.
Staphylococcus aureus is resistant to α-defensins, antimicrobial peptides that play an important role in oxygen-independent killing of human neutrophils. The dlt operon mediates d-alanine incorporation into teichoic acids in the staphylococcal cell envelope and is a determinant of defensin resistance. By using S. aureus wild-type (WT) and Dlt− bacteria, the relative contributions of oxygen-dependent and -independent antimicrobial phagocyte components were analyzed. The Dlt− strain was efficiently killed by human neutrophils even in the absence of a functional respiratory burst, whereas the killing of the WT organism was strongly diminished when the respiratory burst was inhibited. Human monocytes, which do not produce defensins, inactivated the WT and Dlt− bacteria with similar efficiencies. In addition, mice injected with the Dlt− strain had significantly lower rates of sepsis and septic arthritis and fewer bacteria in the kidneys, compared with mice infected with the WT strain
This chapter characterizes the secretion kinetics of putative virulence factors of Legionella pneumophila in comparison with those of non-L. pneumophila species during the phase of growth, when nutritional factors may become limited. To investigate the secretion pattern of different Legionella species, culture supernatants were evaluated for acid phosphatase, protease, phospholipase A (PLA), and lysophospholipase A (LPLA) activities. The experiments revealed that the examined enzyme activities seem to be secreted throughout the whole Legionella genus, except L. longbeachae and L. micdadei lack the majority of the activities tested. Differences in PLA secretion of different isolates within one Legionella species (L. steigerwaltii) have been described. L. gormanii, L. steigerwaltii, and L. pneumophila exhibited the highest PLA activity, and both L. gormanii and one of the tested L. pneumophila strains exhibited the most prominent LPLA activity. To estimate the amount of lysophosphatidylcholine (LPC) generated from culture supernatants of different Legionella species, the authors incubated culture supernatants with dipalmitoylphosphatidylchohne (DPPC) and analyzed the lipids by thin-layer chromatography. Culture supernatants from the exponential growth phase of both L. pneumophila and L. steigerwaltii generated considerable amounts of LPC. LPC was also produced by L. gormanii but not before bacteria entered the stationary phase.
ABSTRACT Bacterial phospholipases are regarded as a major virulence factor in infection. In bacteria associated with pneumonia, destruction of lung surfactant and host cell membranes by bacterial phospholipases secreted during infection is thought to contribute to the disease. Phospholipase C (PLC) activity has been described in several Legionella species (W. B. Baine, J. Gen. Microbiol. 134:489–498, 1988; W. B. Baine, J. Gen. Microbiol. 131:1383–1391, 1985). By using detection methods such as thin-layer chromatography and mass spectrometry, PLC activity could not be detected in several strains of Legionella pneumophila . Instead, phospholipid degradation was identified to be caused by a novel PLA activity. We could demonstrate that PLA secretion starts at the mid-exponential-growth phase when bacteria were grown in liquid culture. Several Legionella species secreted different amounts of PLA. Legionella PLA may act as a powerful agent in the mediation of pathogenicity due to destruction of lung surfactant and epithelial cells.
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