Tomas G. Kloosterman

The cop operon is required for copper homeostasis and contributes to virulence in Streptococcus pneumoniae

High levels of copper are toxic and therefore bacteria must limit free intracellular levels to prevent cellular damage. In this study, we show that a number of pneumococcal genes are differentially regulated by copper, including an operon encoding a CopY regulator, a protein of unknown function (CupA) and a P1‐type ATPase, CopA, which is conserved in all sequenced Streptococcus pneumoniae strains. Transcriptional analysis demonstrated that the cop operon is induced by copper in vitro, repressed by the addition of zinc and is autoregulated by the copper‐responsive CopY repressor protein. We also demonstrate that the CopA ATPase is a major pneumococcal copper resistance mechanism and provide the first evidence that the CupA protein plays a role in copper resistance. Our results also show that copper homeostasis is important for pneumococcal virulence as the expression of the cop operon is induced in the lungs and nasopharynx of intranasally infected mice, and a copA‐ mutant strain, which had decreased growth in high levels of copper in vitro, showed reduced virulence in a mouse model of pneumococcal pneumonia. Furthermore, using the copA‐ mutant we observed for the first time in any bacteria that copper homeostasis also appears to be required for survival in the nasopharynx.

The novel transcriptional regulator SczA mediates protection against Zn2+ stress by activation of the Zn2+‐resistance gene czcD in Streptococcus pneumoniae

Maintenance of the intracellular homeostasis of metal ions is important for the virulence of many bacterial pathogens. Here, we demonstrate that the czcD gene of the human pathogen Streptococcus pneumoniae is involved in resistance against Zn2+, and that its transcription is induced by the transition‐metal ions Zn2+, Co2+ and Ni2+. Upstream of czcD a gene was identified, encoding a novel TetR family regulator, SczA, that is responsible for the metal ion‐dependent activation of czcD expression. Transcriptome analyses revealed that in a sczA mutant expression of czcD, a gene encoding a MerR‐family transcriptional regulator and a gene encoding a zinc‐containing alcohol dehydrogenase (adhB) were downregulated. Activation of the czcD promoter by SczA is shown to proceed by Zn2+‐dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. This is the first example of a metalloregulatory protein belonging to the TetR family that has been described. The presence in S. pneumoniae of the Zn2+‐resistance system characterized in this study might reflect the need for adjustment to a fluctuating Zn2+ pool encountered by this pathogen during infection of the human body.

Regulation of Glutamine and Glutamate Metabolism by GlnR and GlnA in Streptococcus pneumoniae

Several genes involved in nitrogen metabolism are known to contribute to the virulence of pathogenic bacteria. Here, we studied the function of the nitrogen regulatory protein GlnR in the Gram-positive human pathogen Streptococcus pneumoniae. We demonstrate that GlnR mediates transcriptional repression of genes involved in glutamine synthesis and uptake (glnA and glnPQ), glutamate synthesis (gdhA), and the gene encoding the pentose phosphate pathway enzyme Zwf, which forms an operon with glnPQ. Moreover, the expression of gdhA is also repressed by the pleiotropic regulator CodY. The GlnR-dependent regulation occurs through a conserved operator sequence and is responsive to the concentration of glutamate, glutamine, and ammonium in the growth medium. By means of in vitro binding studies and transcriptional analyses, we show that the regulatory function of GlnR is dependent on GlnA. Mutants of glnA and glnP displayed significantly reduced adhesion to Detroit 562 human pharyngeal epithelial cells, suggesting a role for these genes in the colonization of the host by S. pneumoniae. Thus, our results provide a thorough insight into the regulation of glutamine and glutamate metabolism of S. pneumoniae mediated by both GlnR and GlnA.

The cop operon is required for copper homeostasis and contributes to virulence in Streptococcus pneumoniae.

High levels of copper are toxic and therefore bacteria must limit free intracellular levels to prevent cellular damage. In this study, we show that a number of pneumococcal genes are differentially regulated by copper, including an operon encoding a CopY regulator, a protein of unknown function (CupA) and a P1‐type ATPase, CopA, which is conserved in all sequenced Streptococcus pneumoniae strains. Transcriptional analysis demonstrated that the cop operon is induced by copper in vitro, repressed by the addition of zinc and is autoregulated by the copper‐responsive CopY repressor protein. We also demonstrate that the CopA ATPase is a major pneumococcal copper resistance mechanism and provide the first evidence that the CupA protein plays a role in copper resistance. Our results also show that copper homeostasis is important for pneumococcal virulence as the expression of the cop operon is induced in the lungs and nasopharynx of intranasally infected mice, and a copA‐ mutant strain, which had decreased growth in high levels of copper in vitro, showed reduced virulence in a mouse model of pneumococcal pneumonia. Furthermore, using the copA‐ mutant we observed for the first time in any bacteria that copper homeostasis also appears to be required for survival in the nasopharynx.

CcpA ensures optimal metabolic fitness of Streptococcus pneumoniae.

In Gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner.

Opposite Effects of Mn2+ and Zn2+ on PsaR-Mediated Expression of the Virulence Genes pcpA, prtA, and psaBCA of Streptococcus pneumoniae

Homeostasis of Zn(2+) and Mn(2+) is important for the physiology and virulence of the human pathogen Streptococcus pneumoniae. Here, transcriptome analysis was used to determine the response of S. pneumoniae D39 to a high concentration of Zn(2+). Interestingly, virulence genes encoding the choline binding protein PcpA, the extracellular serine protease PrtA, and the Mn(2+) uptake system PsaBC(A) were strongly upregulated in the presence of Zn(2+). Using random mutagenesis, a previously described Mn(2+)-responsive transcriptional repressor, PsaR, was found to mediate the observed Zn(2+)-dependent derepression. In addition, PsaR is also responsible for the Mn(2+)-dependent repression of these genes. Subsequently, we investigated how these opposite effects are mediated by the same regulator. In vitro binding of purified PsaR to the prtA, pcpA, and psaB promoters was stimulated by Mn(2+), whereas Zn(2+) destroyed the interaction of PsaR with its target promoters. Mutational analysis of the pcpA promoter demonstrated the presence of a PsaR operator that mediates the transcriptional effects. In conclusion, PsaR is responsible for the counteracting effects of Mn(2+) and Zn(2+) on the expression of several virulence genes in S. pneumoniae, suggesting that the ratio of these metal ions exerts an important influence on pneumococcal pathogenesis.

Transcriptional response of Streptococcus pneumoniae to Zn2+ limitation and the repressor/activator function of AdcR

Zinc (Zn(2+)) is an important trace metal ion that has been shown to regulate the expression of several (virulence) genes in streptococci. Previously, we analyzed the genome-wide response of S. pneumoniae to Zn(2+)-stress. In this work, we have performed a transcriptomic analysis to identify genes that are differentially expressed under intracellular Zn(2+) limitation. This revealed a number of genes that are highly upregulated in the absence of extracellular Zn(2+), amongst which the genes belonging to the regulon of the Zn(2+)-responsive repressor AdcR, like adcBCA, encoding a Zn(2+)-dependent ABC-uptake system, adcAII, encoding a Zn(2+)-binding lipoprotein, and also virulence genes belonging to the Pht family (phtA, phtB, phtD and phtE). Using transcriptome analysis, lacZ-reporter studies, in vitro DNA binding experiments, and in silico operator predictions, we show that AdcR directly represses the promoters of adcRCBA, adcAII-phtD, phtA, phtB and phtE in the presence of Zn(2+). AdcR can also function as an activator, since in the presence of Zn(2+) it directly induces expression of adh that encodes a Zn(2+)-containing alcohol dehydrogenase. In conclusion, the genome-wide transcriptional response of S. pneumoniae to Zn(2+) limitation was established, which is mainly mediated via direct regulation by the Zn(2+)-dependent regulator AdcR.

Search for Genes Essential for Pneumococcal Transformation: the RadA DNA Repair Protein Plays a Role in Genomic Recombination of Donor DNA

We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

Site-specific contributions of glutamine-dependent regulator GlnR and GlnR-regulated genes to virulence of Streptococcus pneumoniae

ABSTRACT The transcriptional regulator GlnR of Streptococcus pneumoniae is involved in the regulation of glutamine and glutamate metabolism, controlling the expression of the glnRA and glnPQ-zwf operons, as well as the gdhA gene. To assess the contribution of the GlnR regulon to virulence, D39 wild-type and mutant strains lacking genes of this regulon were tested in an in vitro adherence assay and murine infection models. All of the mutants, except the ΔglnR mutant, were attenuated in adherence to human pharyngeal epithelial Detroit 562 cells, suggesting a contribution of these genes to adherence during the colonization of humans. During murine colonization, only the ΔglnA mutant and the glnP-glnA double mutant (ΔglnAP) were attenuated, in contrast to ΔglnP, indicating that the effect is caused by the lack of GlnA expression. In our pneumonia model, only ΔglnP and ΔglnAP showed a significantly reduced number of bacteria in the lungs and blood, indicating that GlnP is required for survival in the lungs and possibly for dissemination to the blood. In intravenously infected mice, glnP and glnA were individually dispensable for survival in the blood whereas the ΔglnAP mutant was avirulent. Finally, transcriptome analysis of the ΔglnAP mutant showed that many genes involved in amino acid metabolism were upregulated. This signifies the importance of glutamine/glutamate uptake and synthesis for full bacterial fitness and virulence. In conclusion, several genes of the GlnR regulon are required at different sites during pathogenesis, with glnA contributing to colonization and survival in the blood and glnP important for survival in the lungs and, possibly, efficient transition from the lungs to the blood.

Development of genomic array footprinting for identification of conditionally essential genes in Streptococcus pneumoniae.

ABSTRACT Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput, genome-wide technique, genomic array footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in nonreproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1 showed that both have an equal degree of saturation but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for surviving zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high-zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.