The form and quantity of iron in humans varies significantly at different anatomical locations and it is likely that bacterial pathogens sense these differences, among other signals, and regulate gene expression in response. The exact mechanisms of iron acquisition and regulation in the pneumococcus are still largely unknown. However, the ability of this pathogen to colonize the highly iron-restricted environment of the nasopharynx and also cause invasive diseases in relatively iron-rich sites suggests that iron may be an important environmental signal for gene regulation.
A signature-tagged mutagenesis study in type 3 pneumococcus suggested a role for smrB
(iron-dependent regulator) in pneumococcal virulence 
. Although the authors proposed the gene designation smrB
, we suggest the more associative idtr
nomenclature. This gene was conserved in various unrelated pneumococcal strains and capsule types (data not shown).
We did not detect any significant difference in growth between wild-type and mutant either in presence or absence of iron in vitro. Additionally, no differences were observed between the mutant and wild-type in their ability to utilize a variety of iron sources (data not shown). The mutant forms clusters and aggregates in both the presence and absence of iron. These observations suggest that idtr has no significant role during pneumococcal growth in vitro but in some way affects bacterial cell-cell adhesion or daughter cell separation during cell division. TIGR4 and Δidtr did not differ significantly in growth rates in blood following bacteremia up to 48 hours after infection. In relatively iron-rich environments such as blood idtr is not critical for pneumococcal growth. This observation parallels that seen in vitro in which the mutant was able to replicate as well as wild-type in presence of high iron concentration.
The contribution of idtr to pneumococcal sepsis was evaluated using a mouse model and both intravenous and intranasal inoculation. The Δidtr mutant was significantly attenuated in the sepsis model by both routes of infection as compared to the parent strain but the more striking difference was observed with the intransal route of infection. We postulate that idtr is essential specifically during transition from the nasopharyngeal mucosa to submucosal tissue and blood. The Δidtr mutant could be isolated from the nasopharynx two days after inoculation but not after day five, so lack of idtr may result in an even earlier deficiency, that is, an inability to efficiently colonize the nasopharynx. In either case it is likely that gene regulation by idtr is critical at mucosal surfaces where the concentration of extracellular iron in any form is exceedingly low.
Because increased mortality in mice infected with TIGR4 strain was not the result of more rapid cell growth in vivo, we selected ten known and putative virulence genes which might potentially be directly or indirectly regulated by idtr
. We had previously studied these same genes in TIGR4 and found that they are differentially regulated in different anatomic sites in mouse models 
. The expression of the selected genes was not markedly different between wild-type and the mutant in vitro but pronounced differences were noted during growth in vivo. Gene expression in Δidtr
was increased compared with wild-type in nasopharyngeal colonization and pneumonia, and was effectively unchanged during bacteremia for all genes except hemolysin. These results suggest that idtr
does play a role in modulation of pneumococcal virulence. Based on these results we hypothesize that idtr
contributes to repression of certain pneumococcal virulence-associated genes at mucosal surfaces and is de-repressed during bacteremia, possibly as a function of iron availability. An iron-dependent transcriptional regulator has been previously associated with virulence in a type 3 strain in pneumonia and bacteremia models by signature-tagged mutagenesis 
. This study extends these findings to nasopharyngeal colonization and suggests that iron may be an important signal with effects on genes involved with virulence.
Sepsis results from systemic infection and the resultant systemic inflammatory responses 
. The innate immune responses are critical inducers of sepsis syndrome in response to bacterial products and cellular components. Cytokines play a central role in regulation of the innate immune response and, therefore, in the manifestation of sepsis 
. An exaggerated pro-inflammatory response which is the hall mark of sepsis is associated with high mortality both in humans and animal models. To uncover possible reasons for the improved survival of mice infected with Δidtr
we evaluated the host cytokine response. The concentration of 14 cytokines known to play an important role in invasive pneumococcal disease was evaluated in plasma and was found to be significantly decreased in plasma samples obtained from mice infected with Δidtr
as compared to TIGR4 infected mice. Most of the cytokines (Eotaxin, G-CSF, IFN-γ, IL-1β, IL-17, MIP-2, KC, MIP-1α, RANTES, TNF-α, IL-12, MCP-1) that were tested are pro-inflammatory cytokines except for IL-10 which is anti- inflammatory and IL-6 which has both pro 
and anti- inflammatory effects 
. Recent evidence indicates that both pro and anti- inflammatory responses are simultaneously regulated even in early stages of sepsis 
. Increased levels of all the cytokines tested are associated with a poor prognosis in sepsis patients or animal models of sepsis 
. Combined high levels of IL-10 and IL-6 are associated with a very high risk of death in sepsis patients 
This difference was not related to a faster growth rate and higher bacterial burden with TIGR4, as both wild-type and mutant were at the same approximate density in the blood at the time of cytokine sampling. These results imply that idtr
not only modulates the bacterial virulence but also modulates the host response to pneumococcal infection. The mechanisms by which this modulation occurs remain to be determined. It is likely that idtr
controls genes which encode pneumococcal surface-exposed components or other factors which interact with the host immune system. To our knowledge, this is the first report indicating a role of iron dependent transcription regulator in host immune response to pneumococcal infections. The role of iron-regulated bacterial genes in modulation of host responses has been reported for other Gram-positive pathogens. In Staphylococcus aureus
the inactivation of fur
is reported to be associated with increased nitric oxide sensitivity 
. In Mycobacterium smegmatis
, insertional inactivation of ideR
(a homolog of dtxR
) was shown to decrease production of manganese superoxide dismutase and catalase/peroxidase (katG), and increase susceptibility to killing by H2
IDTR has an important role in virulence and gene expression and its function is likely related to the form and quantity of available iron at different anatomic sites of the host. Invasive disease in humans follows translocation of pneumococci from mucosal surfaces of the nasopharynx to the lower respiratory tract and, in some cases, dissemination via blood. Environmental conditions are markedly different at each location and the concentrations of certain nutrients necessary for pneumococcal growth almost certainly function, by various pathways, to regulate bacterial gene expression. Future work will define the role of IDTR on global protein expression both in vitro and within a host and undoubtedly expand our understanding the complete subset of genes which are controlled either directly or indirectly by IDTR. Many of these gene products interact with host immune cells and contribute to pro-inflammatory cytokine responses and subsequent mortality in murine models. The identification of these bacterial gene products, and their specific interactions with the host immune system, will allow greater understanding of the pathogenesis of invasive pneumococcal infections and identify potential points at which intervention may be possible to reduce morbidity and mortality.