点击显示 收起
Veterans Affairs Medical Center, Research Service
Departments of Surgery and of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee
The role played by host-pathogen interactions in regulation of expression of streptococcal virulence factors in vivo is beginning to become clear. We have reported that the expression of 2 streptococcal virulence factors, the streptococcal pyrogenic exotoxin (Spe) A and the cysteine protease SpeB, was reciprocally modulated during infection with Streptococcus pyogenes. To identify host signals mediating this reciprocal regulation, we cocultured clonal M1T1 isolates with human peripheral blood mononuclear cells (PBMCs). In accordance with our in vivo findings, when bacteria were in direct contact with human PBMCs or were separated in transwells, expression of speA was induced, whereas expression of speB was down-regulated. This phenomenon was mediated by transferrin and lactoferrin and was influenced by the iron-saturation status of these proteins. Iron chelation from media induced expression of speA, but to a much lesser degree than did that with apotransferrin and lactoferrin, suggesting additional effects of these ferrins on modulation of expression of speA and speB. Thus, ferrins may play an important role in host-pathogen interactions in skin and mucosal tissues.
Group A streptococci (GAS) are generally responsible for mucosal or skin infections leading to pharyngitis and impetigo. Occasionally, these organisms can also cause severe invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock syndrome [1], or chronic illnesses, such as rheumatic heart disease [2]. Several virulence factors, including the streptococcal pyrogenic exotoxins (Spes), play pivotal roles in invasive GAS diseases. Although several GAS serotypes have been isolated from invasive GAS cases, the M1T1 serotype has been the serotype most frequently isolated from both invasive and noninvasive GAS cases worldwide. This M1T1 clonal strain harbors speA, speB, speF, speG, speJ, and smeZ [3]; however, previous studies have shown variable expression of these Spe genes among clonal M1T1 isolates, particularly for SpeA and SpeB. SpeA is an important GAS superantigen that contributes to the pathogenesis of invasive disease [2, 46]. SpeB is a cysteine protease that plays a critical yet complex role in disease pathogenesis, because of its ability to modify several bacterial virulence proteins as well as several host proteins (reviewed in [711]). We observed that almost 40% of clinical invasive M1T1 isolates expressed no detectable amount of SpeA [3]. Similar observations were made in several other studies, in which only 20%50% of speA+ GAS strains were found to express SpeA [1215]. Although we could not find a correlation between expression of SpeA and the severity of invasive GAS infection, we found that expression of SpeB was inversely related to the severity of disease among M1T1 isolates [16].
To understand the basis for variable expression of Spe among clonal M1T1 isolates, we developed a mouse model in which expression of speA can be monitored in vivo over time [17]. All M1T1 isolates that originally possessed the SpeB+/SpeA- phenotype switched to a SpeB-/SpeA+ phenotype by day 37 in vivo, suggesting that host signals might have induced this switch. Although expression of SpeA was induced, expression of SpeB was temporally down-regulated and eventually turned off [17]. This reciprocal phenotype was stable when the bacteria were recovered from the mice tissue chambers after day 7, suggesting that this switch in expression of SpeA and SpeB may be advantageous to the bacteriaexpression of SpeA has been shown to increase GAS survival in vivo [5]. Furthermore, we and others have shown that, by turning off expression of SpeB in certain tissues, the bacteria preserve the integrity of several key virulence components, including the surface M protein and several superantigens [16, 18, 19].
In the present study, we set out to identify host signals that may be responsible for modulation of expression of SpeA and SpeB. We investigated whether the same phenomenon could be reproduced ex vivo and, if so, what is the nature of the molecules mediating this effect. We report that M1T1 bacteria cocultured with human peripheral blood mononuclear cells (PBMCs) up-regulated expression of speA and down-regulated expression of speB. Further investigations led to the finding that human apotransferrin and lactoferrin are mediators of this phenomenon.
MATERIALS AND METHODS
Bacterial strains.
We used 2 representative clinical, clonal M1T1 GAS isolates from severe invasive-infection cases [3]. These strains harbor speA, speB, speF, speG, speJ, and smeZ1 but lack speC, speH, speI, and smeZ2 [3]. Both have the SpeB+/SpeA- phenotype in vitro [17]. The bacteria were grown to stationary phase, as described elsewhere [16], were washed in Hanks' balanced salt solution (HBSS) or MEM, and were cultured in defined media, as indicated for each study.
PBMCs and cell lines.
Human PBMCs were isolated from blood from healthy volunteers, as described elsewhere [16]. In some studies, lymphocytes and monocytes were separated [20] and cultured separately with the bacteria. The human Jurkat T lymphoma line and the pharyngeal cell line Detroit 562 were used in certain studies.
Coculture of M1T1 GAS isolates with cultured PBMCs in serum-free media.
PBMCs, Jurkat cells, and pharyngeal cells were cultured overnight in RPMI medium plus 10% fetal calf serum and were washed free of serum, and 107 cells were cocultured with 108 SpeB+/SpeA- bacteria for 14 h in 2 mL of MEM, HBSS, or chemically defined HL-1 medium (Biowhittaker). The culture media were recovered at varying times, filter sterilized, and concentrated by use of Microcone YM-10 concentrators (Millipore). In some experiments, the bacteria were placed in 12-well transwells (Costar), in which they were physically separated from leukocytes by a 0.4-m semipermeable membrane that allows free flow of solutes between 2 compartments. The bacteria were always seeded in the inner well.
Assessment of expression of SpeA and SpeB proteins and genes.
Production of SpeA and SpeB proteins was assessed by Western blotting, as described elsewhere [3], and was quantified by use of a flatbed scanner and BioRad Quantity One software. To quantify Spe transcripts, total RNA was isolated from bacteria, as described elsewhere [18], and was quantified spectrophotometerically. RNA was freed of the contaminating DNA by repeated DNase treatments [18]. For accurate quantification of numbers of transcripts, we generated synthetic RNA standards for the genes of interest. First, DNA templates of genes of interest were generated by use of primers designed to amplify a product larger than the desired PCR product required for real-time quantitative PCR. Primer pairs used for the DNA templates were recA (741bp; 5-gctgctgacgatggtttgtta-3 and 5-ccagccgaacagaagcataga-3), speA (505 bp; 5-gtattgaagaaaatggtattttttg-3 and 5-attcttgagcagttaccattttt-3), and speB (412 bp; 5-agaattgatggctgatgttggta-3 and 5-ctaaggtttgatgcctacaaca-3). The PCR amplicon was ligated into pCR4-TOPO TA Cloning Vector (Invitrogen). The resulting plasmids were propagated in DH5-competent Escherichia coli and were sequenced to check for orientation of inserts. Depending on the orientation of the insert relative to the promoter, the restriction enzymes Not1 (T3 promoter) and Spe1 (T7 promoter) were used to generate a linearized plasmid, which was transcribed into RNA sense strand by use of T3 or T7 RNA polymerase, depending on the orientation of the insert. In vitrosynthesized RNA was purified by use of RNA Stat-60 (Tel-Test), precipitated, suspended in nuclease-free water, quantified, aliquoted, and stored at -80°C.
RNA standards and total RNA from bacteria were converted to cDNA by use of reverse transcriptase and random hexamers. Gene expression was quantified by use of an ABI-PRISM 7900HT sequence detection system with gene-specific primers in PCR buffer containing SYBR Green. The primer sequences were recA (73 bp; 5-cgactgtggctttacatgctgta-3 and 5-tgctcggcatcgataaagg-3), speA (92 bp; 5-ctagatctgtgaagttggcttgga-3 and 5-ttttttgttttagtgacatttcttgga-3), and speB (69 bp; 5-cgcactaaacccttcagctctt-3 and 5-acagcactttggtaaccgttga-3).
Stripping and saturation of transferrin with iron.
Human apotransferrin (Sigma) was sequentially saturated with iron by incubation with FeCl3, as described elsewhere [21]. A 200-mol/L solution of apotransferrin was prepared in 40 mmol/L Tris/20 mmol/L NaHCO3 buffer (pH 7.4) and saturated with iron, to 30%, 60%, and 100%, by adding 120, 240, and 400 mol/L FeCl3, respectively, and incubating for 1 h at 37°C. Unbound iron was removed by overnight dialysis using a Slide-a Lyzer dialysis cassette (cutoff, 10 kDa; Pierce) against 2 changes of 40 mmol/L Tris/20 mmol/L NaHCO3 buffer (pH 7.4). The proteins were filter sterilized and saved for use in the coculture assays. Human holotransferrin (Sigma) was stripped of iron by dialysis against 0.1 mol/L sodium citrate buffer (pH 4.5) [21]. The stripping of holotransferrin and iron-saturation levels of apotransferrin were confirmed by determining the total iron-binding capacity of holotransferrin and of apotransferrin, by use of an Iron/TIBC kit from Teco Diagnostics.
RESULTS
Induction of expression of SpeA after coculture of SpeA- bacteria with PBMCs.
When a representative SpeB+/SpeA- isolate of the M1T1 clone was cocultured with resting human PBMCs in MEM, a marked induction in expression of SpeA was seen as early as 1 h after coculture, with no further increase after 4 h (figure 1A). The activation of PBMCs by the polyclonal mitogen phytohemagglutinin (PHA) did not cause further induction of expression of SpeA (figure 1B). To determine which cells in the PBMC population provided signals for modulation of Spe, we fractionated enriched T cells from B cells and monocyte populations before coincubating them with the SpeB+/SpeA- bacteria. Expression of SpeA was induced in the presence of all cell populations tested, including Jurkat (figure 1C) and Detroit 562 (data not shown).
Our earlier observation that expression of SpeA and that of SpeB were temporally yet reciprocally regulated prompted us to look at expression of SpeB as well. Compared with a more consistent up-regulation of expression of SpeA, expression of SpeB was down-regulated to various levels (214-fold) after coculture with PBMCs. The mean (±SD) fold increase in expression of SpeA from 5 independent experiments was 16 (±6), whereas the mean (±SD) fold decrease in expression of SpeB from the same experiments was 8 (±5.3). This wide variation in the degree of down-regulation of expression of SpeB can be attributed mainly to the presence of residual premade SpeB protein in the bacteria. In fact, when SpeB transcripts were quantified, a 1540-fold decrease in the number of speB transcripts was seen, and only a 210-fold increase in the number of speA transcripts was seen after the 4-h coculture of M1T1 bacteria with PBMCs (figure 2). However, as will be explained below, the number of SpeA transcripts was significantly higher when assessed at earlier time points. Thus, in the present study, the amount of SpeA protein and the number of SpeB transcripts were more reliable measures of their reciprocal expressions.
No requirement for physical contact between PBMCs and bacteria for induction of expression of SpeA.
When PBMCs and SpeB+/SpeA- bacteria were separated in a transwell by a 0.4-m semipermeable membrane, induction of expression of SpeA occurred regardless of whether the bacteria were in physical contact with the PBMCs or were separated from the PBMCs by the semipermeable membrane (figure 3); however, the mean increase in expression of SpeA in bacteria cultured in transwells was only 33.7% ± 10% of that seen when the bacteria were in direct contact with the PBMCs. Induction of expression of SpeA was also seen when either media from resting or PHA-stimulated PBMCs cultured without bacteria or media from PBMCs stimulated with a speA mutant of the parent strain were added to the SpeB+/SpeA- bacteria; however, similar to what was seen in the transwells, under these conditions, the mean fold increase in production of SpeA was only 20% ± 9% of that seen when the bacteria were in direct contact with the PBMCs. Together, the data suggest that direct PBMC-bacteria interactions are required for optimal production of SpeA but that the molecules responsible for this induction are soluble, secreted, and diffusible.
Identification of human transferrin and lactoferrin as inducers of SpeA.
The only proteins in the 10-kDa fraction of HL-1 medium were human apotransferrin and insulin, so we tested each of them separately and in combination. The addition of apotransferrin to bacteria cultured in MEM caused marked induction of expression of SpeA, in a dose-dependent manner. Insulin, on the other hand, failed to induce expression of SpeA and did not further increase the levels of expression of SpeA induced by apotransferrin (figure 5A and 5B). The induction of expression of SpeA was also confirmed at the transcriptional level. The increase in the number of SpeA transcripts was first observed at 30 min after addition of apotransferrin, reached a peak increase of 7-fold at 60 min, and declined thereafter (table 1). In the absence of apotransferrin, the number of SpeA transcripts remained unaltered over the course of the 2-h incubation. The fold increases in SpeA-specific transcripts after culture of SpeA- bacteria in media alone (without apotransferrin) were 1, 1.16, 1.13, and 1.27 at 0, 30, 60, and 120 min, respectively.
Iron-saturation statusdependent ability of transferrin to induce expression of SpeA.
To determine whether the effect of transferrin was through scavenging free iron from the medium, induction of expression of SpeA was compared in the presence of holotransferrin before and after stripping of iron. Induction of expression of SpeA was additionally compared in the presence of apotransferrin before and after sequential saturation. Expression of SpeA was induced in bacteria cultured in MEM in the presence of either apotransferrin or iron-stripped holotransferrin (figure 7A). By contrast, the presence of holotransferrin resulted in a significantly lower induction of expression of SpeA. Sequential saturation of apotransferrin with iron resulted in a sequential decrease in its ability to induce up-regulation of expression of SpeA (figure 7B).
Together, the data suggest that the effect of transferrin and lactoferrin on induction of expression of SpeA may be through their iron-scavenging properties and that iron deprivation may induce a stress signal that reciprocally modulates the expression of speA and speB. To further investigate the effect of iron deprivation, we treated MEM or HL-1 medium with a strong iron chelator, deferoxamine mesylate, at concentrations of 0.25, 0.5. and 1 mmol/L. When the SpeB+/SpeA- bacteria were cultured in deferoxamine-treated MEM or HL-1, there was a noticeable increase in production of speA (data not shown). However, this increase was less marked, compared with that seen in the presence of apotransferrin. These observations suggest that, in addition to causing iron deprivation (which leads to stress signals), transferrin might also affect the regulation of expression of superantigen in a trans manner, through yet another unidentified intermediate.
DISCUSSION
GAS cause human diseases by adapting to various niches that they encounter in human hosts. The ability of GAS to be able to cause diseases of varying severities depends on the response of regulatory elements to environmental signals and their control over the expression of virulence factors. Environmental factorssuch as osmolarity, temperature, CO2, and restricted ironhave been shown to regulate the expression of GAS virulence factors [2326]. We previously showed that expression of SpeA and SpeB is influenced by host environmental signals and reported that SpeB+/SpeA- isolates shift to a SpeB-/SpeA+ phenotype after inoculation in mouse tissue chambers [17]. Here, we have shown that induction of expression of speA and down-regulation of expression of speB can be effected ex vivo by coculturing SpeB+/SpeA- isolates with human PBMCs. We have further demonstrated that this reciprocal expression of spe genes is mediated by human transferrin and lactoferrin.
Several groups have reported induction of expression of spe genes after coincubation of bacteria with human cells. Broudy et al. reported up-regulation of expression of phage-encoded SpeC and DNAse after coculture with human pharyngeal cells and found this phenomenon to be associated with induction of phage [27, 28]. A similar observation was made by Banks et al. [29] in M3 GAS, and Voyich et al. [30] showed that several GAS prophage and chromosomal genes involved in virulence, oxidative stress, cell-wall biosynthesis, and gene regulation were up-regulated during phagocytic interaction with human polymorphonuclear lymphocytes. However, the nature of the signals provided by mammalian cells that induce a change in expression of GAS genes was not determined in the studies cited above.
The discovery, here, that transferrin and lactoferrin are capable of inducing Spe phenotype switch in M1T1 bacteria was made as a result of our using chemically defined medium that contained apotransferrin. The induction of expression of SpeA and down-regulation of expression of SpeB by transferrin and lactoferrin took place within minutes of coincubation with the bacteria and was seen both at the RNA and the protein levels, suggesting that the signal provided by these ferrins reciprocally affects the transcription of speA and speB. Another, mutually nonexclusive possibility is that these ferrins induce the phage that carries speA, resulting in increased expression of SpeA. However, SpeB is chromosomally encoded, and previous studies have shown that induction of phage-encoded Spe could occur without induction of phage. Whereas Brody et al. showed that expression of SpeC was accompanied by induction of phage [27], Banks et al. showed that up-regulation of expression of SpeA occurred without induction of prophage, which encodes it [29]. We observed only a partial induction of SpeA prophage after coculture with PBMCs. Thus, it is likely that, in addition to an effect on induction of phage, these ferrins may also regulate the transcription of speA and speB indirectly.
We believe that the effect of transferrin and lactoferrin on expression of Spe genes is physiologically important because the concentrations used in the present study were orders of magnitude below those found in vivo, which can be in the range of 24 mg/mL for serum transferrin and up to 14 mg/mL for lactoferrin at mucosal sites, such as skin and saliva [22]. GAS is more likely to interact with lactoferrin on mucosal surfaces, but, once the bacteria invade and become blood borne, transferrin is more likely to affect modulation of Spe genes.
The mode by which ferrins modulate the expression of GAS genes is not entirely clear. We found that the ability of transferrin and lactoferrin to modulate the expression of SpeA and SpeB was largely dependent on the iron-saturation status of these proteins, suggesting that the scavenging of iron may be a stress signal that induces expression of SpeA. These ferrins belong to a family of iron-binding proteins that are instrumental in iron transport in mammals and scavenge iron from serum or other biological fluids, thus depriving microorganisms of iron, inhibiting microbial growth, and generating other microbiostatic defense mechanisms in the host. Our attempts to reproduce the phenomenon of reciprocal expression of Spe by chelating iron through addition of escalating amounts of deferoxamine did not have as strong an effect on induction of expression of speA gene, compared with that seen when apotransferrin or lactoferrin was added. These results, together with the observation that low levels of expression of SpeA were induced in the presence of holotransferrin, suggest that, although iron chelation by these ferrins is likely to play a major role in modulation of expression of Spe genes, transferrin and lactoferrin may also have additional effects on expression of these genes, effects that are unrelated to iron deprivation.
To our knowledge, Streptococcus pyogenes has not been shown to use holotransferrin or lactoferrin as a source of iron. Our attempts to identify GAS receptors for these proteins by direct binding were unsuccessful. This does not rule out transient or low-affinity binding, which might have taken place to a level that was sufficient to induce signals in the bacteria and that led to the modulation of transcription of Spe genes. Unlike several gram-negative bacteria, which express outer-membrane receptors that directly bind to transferrin and lactoferrin [3133], the receptors for these proteins on gram-positive bacteria have not been characterized. The only transferrin receptor among gram-positive bacteria that has been characterized is a cell surfaceassociated glyceraldehyde-3-phosphate dehydrogenate of Staphylococcus aureus [34]. This observation was later refuted, and another cell wallanchored protein, StbA, was reported to be the receptor for transferrin [35]. Blast-homology searches of the genomic database of M1GAS SF370 showed no shared sequences or significant homology with staphylococcal StbA. Our future studies will investigate, in greater depth, the mechanism by which the ferrins modulate the transcription of Spe genes, including the mechanism by which iron deprivation regulates expression of Spe genes.
The effect of iron on the regulation of gene expression has been shown in several bacterial species, including Brucella, Niesseria, and Mycobacteria [3639]. Diphtheria toxin, a phage-encoded toxin, has been shown to be regulated through the transcriptional repressor DtxR, such that, when bound to iron, it represses production of diphtheria toxin by binding to an operator overlapping the toxin gene promoter [40, 41].
The present study has identified ferrins as host players in the complex host-pathogen interactions that regulate the expression of virulence factors of GAS and affect their ability to cause diseases of varying severity. Although neither expression of SpeA nor up-regulation of expression of SpeA is an isolated event during the host-pathogen interaction, neither is SpeA the only virulence factor produced by the invading bacteria. Instead, the outcome of infection is the result of a complex networking of global and individual regulators, which, in turn, can be affected by the host signals. The down-regulation of expression of speB gene after coculture is supported by our earlier in vivo findings and by those of Raeder et al., who reported that the passage of a SpeB-producing GAS in human blood or in mice selects for a stable, phase-varied GAS with reduced expression of SpeB [19]. The down-regulation of expression of SpeB in blood can help bacteria to evade phagocytosis by keeping its M protein intact, whereas down-regulation of expression of SpeB at the mucosal surface can help to achieve better colonization by enhancing the fibronectin-dependent uptake by the host cells [16]. Thus, a better understanding of the factor(s) affecting the expression of various virulence factors can help us to define the role that differential in vivo and in vitro regulation of gene expression plays in disease progression.
References
1. Low DE, Schwartz B, McGeer A. The reemergence of severe group A streptococcal disease: an evolutionary perspective. In: Scheld WM, Armstrong D, Hughes JM, eds. Emerging infections. Vol. 1. Washington, DC: American Society for Microbiology Press, 1997:93123. First citation in article
2. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000; 13:470511. First citation in article
3. Chatellier S, Ihendyane N, Kansal RG, et al. Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases. Infect Immun 2000; 68:352334. First citation in article
4. Kotb M. Bacterial pyrogenic exotoxins as superantigens. Clin Microbiol Rev 1995; 8:41126. First citation in article
5. Welcher BC, Carra JH, DaSilva L, et al. Lethal shock induced by streptococcal pyrogenic exotoxin A in mice transgenic for human leukocyte antigen-DQ8 and human CD4 receptors: implications for development of vaccines and therapeutics. J Infect Dis 2002; 186:50110. First citation in article
6. Cleary PP, McLandsborough L, Ikeda L, Cue D, Krawczak J, Lam H. High-frequency intracellular infection and erythrogenic toxin A expression undergo phase variation in M1 group A streptococci. Mol Microbiol 1998; 28:15767. First citation in article
7. Norrby-Teglund A, Kotb M. Host-microbe interactions in the pathogenesis of invasive group A streptococcal infections. J Med Microbiol 2000; 49:84952. First citation in article
8. Ashbaugh CD, Wessels MR. Absence of a cysteine protease effect on bacterial virulence in two murine models of human invasive group A streptococcal infection. Infect Immun 2001; 69:66838. First citation in article
9. Collin M, Olsen A. Extracellular enzymes with immunomodulating activities: variations on a theme in Streptococcus pyogenes. Infect Immun 2003; 71:298392. First citation in article
10. Saouda M, Wu W, Conran P, Boyle MD. Streptococcal pyrogenic exotoxin B enhances tissue damage initiated by other Streptococcus pyogenes products. J Infect Dis 2001; 184:72331. First citation in article
11. Rasmussen M, Bjorck L. Proteolysis and its regulation at the surface of Streptococcus pyogenes. Mol Microbiol 2002; 43:53744. First citation in article
12. Chaussee MS, Liu J, Stevens DL, Ferretti JJ. Genetic and phenotypic diversity among isolates of Streptococcus pyogenes from invasive infections. J Infect Dis 1996; 173:9018. First citation in article
13. Holm SE, Norrby A, Bergholm AM, Norgren M. Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 19881989. J Infect Dis 1992; 166:317. First citation in article
14. Nakashima K, Ichiyama S, Iinuma Y, et al. A clinical and bacteriologic investigation of invasive streptococcal infections in Japan on the basis of serotypes, toxin production, and genomic DNA fingerprints. Clin Infect Dis 1997; 25:2606. First citation in article
15. Talkington D, Schwartz B, Black C, et al. Association of phenotypic and genotypic characteristics of invasive Streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome. Infect Immun 1993; 61:336974. First citation in article
16. Kansal RG, Nizet V, Jeng A, Chuang WJ, Kotb M. Selective modulation of superantigen-induced responses by streptococcal cysteine protease. J Infect Dis 2003; 187:398407. First citation in article
17. Kazmi SU, Kansal R, Aziz RK, et al. Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo. Infect Immun 2001; 69:498895. First citation in article
18. Aziz RK, Pabst MJ, Jeng A, et al. Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB. Mol Microbiol 2004; 51:12334. First citation in article
19. Raeder R, Harokopakis E, Hollingshead S, Boyle MD. Absence of SpeB production in virulent large capsular forms of group A streptococcal strain 64. Infect Immun 2000; 68:74451. First citation in article
20. Kotb M, Norrby-Teglund A, McGeer A, et al. An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections. Nat Med 2002; 8:1398404. First citation in article
21. Xiao R, Kisaalita WS. Iron acquisition from transferrin and lactoferrin by Pseudomonas aeruginosa pyoverdin. Microbiology 1997; 143:250915. First citation in article
22. Miehlke S, Reddy R, Osato MS, Ward PP, Conneely OM, Graham DY. Direct activity of recombinant human lactoferrin against Helicobacter pylori. J Clin Microbiol 1996; 34:25934. First citation in article
23. Caparon MG, Geist RT, Perez-Casal J, Scott JR. Environmental regulation of virulence in group A streptococci: transcription of the gene encoding M protein is stimulated by carbon dioxide. J Bacteriol 1992; 174:5693701. First citation in article
24. McIver KS, Heath AS, Scott JR. Regulation of virulence by environmental signals in group A streptococci: influence of osmolarity, temperature, gas exchange, and iron limitation on emm transcription. Infect Immun 1995; 63:45402. First citation in article
25. Smoot JC, Barbian KD, Van Gompel JJ, et al. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc Natl Acad Sci USA 2002; 99:466873. First citation in article
26. Dalton TL, Scott JR. CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J Bacteriol 2004; 186:392837. First citation in article
27. Broudy TB, Pancholi V, Fischetti VA. Induction of lysogenic bacteriophage and phage-associated toxin from group a streptococci during coculture with human pharyngeal cells. Infect Immun 2001; 69:14403. First citation in article
28. Broudy TB, Pancholi V, Fischetti VA. The in vitro interaction of Streptococcus pyogenes with human pharyngeal cells induces a phage-encoded extracellular DNase. Infect Immun 2002; 70:280511. First citation in article
29. Banks DJ, Lei B, Musser JM. Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315. Infect Immun 2003; 71:707986. First citation in article
30. Voyich JM, Sturdevant DE, Braughton KR, et al. Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 2003; 100:19962001. First citation in article
31. Ekins A, Niven DF. Production of transferrin receptors by Histophilus ovis: three of five strains require two signals. Can J Microbiol 2001; 47:41723. First citation in article
32. Evans RW, Oakhill JS. Transferrin-mediated iron acquisition by pathogenic Neisseria. Biochem Soc Trans 2002; 30:7057. First citation in article
33. Bog YS, Andresen LO, Bastholm L, Elling F, Angen O, Heegaard PM. The transferrin receptor of Actinobacillus pleuropneumoniae: quantitation of expression and structural characterization using a peptide-specific monoclonal antibody. Vet Microbiol 2001; 81:5164. First citation in article
34. Modun B, Morrissey J, Williams P. The staphylococcal transferrin receptor: a glycolytic enzyme with novel functions. Trends Microbiol 2000; 8:2317. First citation in article
35. Taylor JM, Heinrichs DE. Transferrin binding in Staphylococcus aureus: involvement of a cell wall-anchored protein. Mol Microbiol 2002; 43:160314. First citation in article
36. Perkins-Balding D, Ratliff-Griffin M, Stojiljkovic I, et al. Iron transport systems in Neisseria meningitidis. Microbiol Mol Biol Rev 2004; 68:15471; table of contents. First citation in article
37. Andrews SC, Robinson AK, Rodriguez-Quinones F. Bacterial iron homeostasis. FEMS Microbiol Rev 2003; 27:21537. First citation in article
38. Dubnau E, Smith I. Mycobacterium tuberculosis gene expression in macrophages. Microbes Infect 2003; 5:62937. First citation in article
39. Roop RM 2nd, Bellaire BH, Valderas MW, Cardelli JA. Adaptation of the Brucellae to their intracellular niche. Mol Microbiol 2004; 52:62130. First citation in article
40. Schmitt MP, Talley BG, Holmes RK. Characterization of lipoprotein IRP1 from Corynebacterium diphtheriae, which is regulated by the diphtheria toxin repressor (DtxR) and iron. Infect Immun 1997; 65:53647. First citation in article
41. Schmitt MP, Holmes RK. Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron. J Bacteriol 1994; 176:11419. First citation in article