SummaryDuring sequencing of an 11.5 kb genomic region of a serotype M49 group A streptococcal (GAS) strain, a series of genes were identified including nra (negative regulator of GAS). Transcriptional analysis of the region revealed that nra was primarily monocistronically transcribed. Polycistronic expression was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra. The deduced Nra protein sequence exhibited 62% homology to the GAS RofA positive regulator. In contrast to RofA, Nra was found to be a negative regulator of its own expression and that of the two adjacent operons by analysis of insertional inactivation mutants. By polymerase chain reaction and hybridization assays of 10 different GAS serotypes, the genomic presence of nra, rofA or both was demonstrated. Nra-regulated genes include the fibronectin-binding protein F2 gene (prtF2) and a novel collagen-binding protein (cpa). The Cpa polypeptide was purified as a recombinant maltose-binding protein fusion and shown to bind type I collagen but not fibronectin. In accordance with nra acting as a negative regulator of prtF2 and cpa, levels of attachment of the nra mutant strain to immobilized collagen and fibronectin was increased above wild-type levels. In addition, nra was also found to regulate negatively (four-to 16-fold) the global positive regulator gene, mga. Using a strain carrying a chromosomally integrated duplication of the nra 3Ј end and an nra -luciferase reporter gene transcriptional fusion, nra expression was observed to reach its maximum during late logarithmic growth phase, while no significant influence of atmospheric conditions could be distinguished clearly.

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death ( P Ͻ 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered ( P Ͻ 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.

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Mitochondrial pseudogenes in the human nuclear genome have been previously described, mostly as a source of artifacts during the analysis of the mitochondrial genome. With the availability of the complete human genome sequence, we performed a comprehensive analysis of mtDNA insertions into the nucleus. We found 612 independent integrations that are evenly distributed among all chromosomes as well as within each individual chromosome. The identified pseudogenes account for a content of at least 0.016% of the human nuclear DNA. Up to 30% of a chromosome's mtDNA pseudogene content is composed of fragments that encompass two or more adjacent mitochondrial genes, and we found no correlation between the abundance of mitochondrial transcripts and the multiplicity of integrations. These observations indicate that the migrations of mitochondrial DNA sequences to the nucleus were predominantly DNA mediated. Phylogenetic analysis of the mtDNA pseudogenes and mtDNA sequences of primates indicate a continuous transfer into the nucleus. Because of the limited window of opportunity for mtDNA transfer to the germline, sperm mtDNA, which is released from degenerating mitochondria after fertilization, could be an important source of nuclear mtDNA pseudogenes.[Online supplemental material available at http://www.genome.org]The presence of DNA in the nucleus, which has a significant homology with mitochondrial DNA (mtDNA), has been known for decades. Examples can be found not only for mtDNA but also for chloroplast DNA. Moreover, these findings have been reported for a variety of species, including more than 60 animal species and plants (for a recent review, see Bensasson et al. 2001). The majority of those nuclear copies were identified, when nuclear DNA was accidentally amplified by PCR, using mtDNA-specific primers to detect mtDNA mutations. Nuclear insertions of mtDNA are also called pseudogenes because those fragments, despite their significant sequence homology, are not transcribed or translated into functional proteins. Part of this is because of the different genetic codes in mitochondrial DNA.

Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.

The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3' end of the core vir regulon. Utilizing the M49 wild type, a mga- mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga- mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome.

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