Francesco Iannelli

Hic, a Novel Surface Protein of Streptococcus pneumoniae That Interferes with Complement Function

The important human pathogen Streptococcus pneumoniae was found to absorb factor H, an inhibitor of complement, from human plasma. We identified the gene encoding a novel surface protein, factor H-binding inhibitor ofcomplement (Hic), in the pspC locus of type 3 pneumococci. Unlike PspC proteins in other serotypes, Hic is anchored to the cell wall by means of an LPXTG motif, and the overall sequence homology to various PspC proteins is low. However, the NH2-terminal region showed significant homology to the NH2-terminal region of several PspC proteins. A fragment of Hic, covering this homologous region, was expressed as a glutathioneS-transferase (GST) fusion protein. GST:Hic39–261 bound radiolabeled factor H and inhibited binding of factor H to pneumococci of different serotypes. Interaction kinetics between GST:Hic39–261 and factor H were studied with surface plasmon resonance and showed a high affinity binding (K A = 5 × 107,K D = 2.3 × 10− 8). Mutant pneumococci lacking Hic showed no absorption of factor H in human plasma and no binding of radiolabeled factor H, suggesting that Hic is responsible for factor H-binding in type 3 pneumococci. Factor H-dependent inhibition of the alternative pathway was not diminished by the presence of GST:Hic39–261. In addition, an intrinsic inhibitory effect of Hic is suggested.

Switch from planktonic to sessile life : a major event in pneumococcal pathogenesis

Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT‐PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence‐specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum‐sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm‐like state.

Allelic variation in the highly polymorphic locus pspC of Streptococcus pneumoniae

PspC, also called SpsA, CbpA, PbcA, and Hic, is a surface protein of Streptococcus pneumoniae studied for its antigenic properties, its capability to bind secretory IgA, C3 and complement factor H, and its activity as an adhesin. In this work we characterized the pspC locus of 43 pneumococcal strains by DNA sequencing of PCR fragments. Using PCR primers designed on two unrelated open reading frames, flanking the pspC locus, it was possible to amplify the pspC locus of each of the 43 strains of S. pneumoniae. In 37 out of 43 strains there was a single copy of the pspC gene, while two tandem copies of pspC were found in the other six strains. The sequence of the pspC locus was different in each of the 43 strains. Insertion sequences were found in the pspC locus of 11 out of 43 strains. Analysis of the deduced amino acid sequence of the PspC variants showed a common organization of the molecules: (i) a 37 amino acid leader peptide which is conserved in all proteins, (ii) an N-terminal portion which is essentially alpha-helical, and is the result of assembly of eight major sequence blocks, (iii) a proline-rich region, and (iv) a C-terminal anchor responsible for the cell surface attachment. By sequence comparison we identified 11 major groups of PspC proteins. Proteins within one group displayed only minor variations of the amino acid sequence. An unexpected finding was that PspC variants could differ in the anchor sequence. While 32 of the PspC proteins displayed the typical choline binding domain of pneumococcal surface proteins, 17 other PspCs showed the LPXTG motif, which is typical of surface proteins of other gram-positive bacteria. This major difference in the anchor region was also observed in the adjacent proline-rich regions which differed considerably in size and composition.

Sialic acid: a preventable signal for pneumococcal biofilm formation, colonization, and invasion of the host.

The correlation between carbohydrate availability, pneumococcal biofilm formation, nasopharyngeal colonization, and invasion of the host has been investigated. Of a series of sugars, only sialic acid (i.e., N-acetylneuraminic acid) enhanced pneumococcal biofilm formation in vitro, at concentrations similar to those of free sialic acid in human saliva. In a murine model of pneumococcal carriage, intranasal inoculation of sialic acid significantly increased pneumococcal counts in the nasopharynx and instigated translocation of pneumococci to the lungs. Competition of both sialic acid-dependent phenotypes was found to be successful when evaluated using the neuraminidase inhibitors DANA (i.e., 2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. The association between levels of free sialic acid on mucosae, pneumococcal colonization, and development of invasive disease shows how a host-derived molecule can influence a colonizing microbe and also highlights a molecular mechanism that explains the epidemiologic correlation between respiratory infections due to neuraminidase-bearing viruses and bacterial pneumonia. The data provide a new paradigm for the role of a host compound in infectious diseases and point to new treatment strategies.

Characterization of a Genetic Element Carrying the Macrolide Efflux Gene mef(A) in Streptococcus pneumoniae

ABSTRACT The mef(A) gene from a clinical isolate ofStreptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames.orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance genemsr(SA).

Upper and Lower Respiratory Tract Infection by Streptococcus pneumoniae Is Affected by Pneumolysin Deficiency and Differences in Capsule Type

ABSTRACT Pneumococci frequently colonize the upper respiratory tract, and these pneumococci are believed to act as a reservoir for infection of the lower respiratory tract and bacteremia. We investigated how the pneumococcal toxin pneumolysin affects the capacity of pneumococci to infect the upper and lower respiratory tract of the mouse. Wild-type Streptococcus pneumoniae serotype 2 and 3 strains, a serotype 2 pneumolysin-deficient mutant, and a serotype 2 mutant with the pneumolysin gene reinserted were used to study differences in colonization and disease. In addition, we also examined a pneumococcal chimeric mutant (capsule type switched from serotype 2 to serotype 3) to gain further insight into the role that capsule plays in nasopharyngeal infection. Absence of pneumolysin was found to be associated with significantly lower numbers of pneumococci in the nasopharynx, trachea, and lungs. Differences in pneumococcal capsule type were found to have significant effects on pneumococcal infection of the nasopharynx, trachea, and lungs. However, it was the combination of capsule type and genetic background that was important, and the influence of this combination varied with the site of infection. For example, in the nasopharynx the wild-type serotype 3 strain and the capsule-switched mutant behaved similarly, whereas in the lungs the mutant that was switched to serotype 3 survived less well than the wild-type serotype 3 strain. The combination of capsule type and genetic background also determined virulence. Thus, the wild-type serotype 3 strain was virulent, whereas the capsule-switched mutant was avirulent.

Pneumococcal zinc metalloproteinase ZmpC cleaves human matrix metalloproteinase 9 and is a virulence factor in experimental pneumonia.

The ZmpC zinc metalloproteinase of Streptococcus pneumoniae, annotated in the type 4 genome as SP0071, was found to cleave human matrix metalloproteinase 9 (MMP‐9). The previously described IgA protease activity was confirmed to be specifically linked to the IgA1‐protease/SP1154 zinc metalloproteinase. MMP‐9 is a protease cleaving extracellular matrix gelatin and collagen and is activated by proteolytic cleavage like most proteases. MMP‐9 is a human protease and is involved in a variety of physiological and pathological matrix degrading processes, including tissue invasion of metastases and opening of the blood–brain barrier. While TIGR4 (serotype 4) and G54 (serotype 19) pneumococcal genome strains have a highly conserved copy of zmpC, the genome of R6 (a derivative of serotype 2 D39 strain) lacks zmpC. Both the analysis for zmpC presence and MMP‐9 cleavage activity in various pneumococcal strains showed correlation of ZmpC with MMP‐9 cleavage activity. When assaying clinical isolates of S. pneumoniae, the zmpC gene was not found in any of the nasal and conjunctival swab isolates, but it was present in 1 out of 13 meningitis isolates and in 6 out of 11 pneumonia isolates. In a murine pneumonia model, infection with a zmpC‐mutant reduced mortality at 3–4 days post‐infection by 75%, when compared with infection with wild‐type strains. These data indicate that the ZmpC pneumococcal protease may play a role in pneumococcal virulence and pathogenicity in the lung.

Streptococcus pneumoniae–Associated Human Macrophage Apoptosis after Bacterial Internalization via Complement and Fcγ Receptors Correlates with Intracellular Bacterial Load

Opsonization enhances Streptococcus pneumoniae-induced human monocyte-derived macrophage (MDM) apoptosis. Both depletion of complement and immunoglobulin from opsonizing serum and blockade of the macrophages CR1, CR3, FcgammaRII, and FcgammaRIII partially decreased MDM apoptosis after S. pneumoniae phagocytosis, and these effects correlated with reduced numbers of internalized bacteria. Chloramphenicol inhibition of protein synthesis by opsonized S. pneumoniae down-regulated subsequent MDM apoptosis. Phagocytosis of an unencapsulated mutant of S. pneumoniae resulted in increased MDM apoptosis, in association with enhanced internalization. Caspase inhibition was associated with decreased killing of bacteria. Enhanced induction of apoptosis by opsonized S. pneumoniae is the result of increased intracellular burden of bacteria, rather than of a specific pattern of engagement of complement receptor or FcgammaR. A dynamic interaction between live intracellular bacteria and the host cell is necessary for induction of apoptosis in MDMs, and induction of apoptosis contributes to the host defense against S. pneumoniae.

Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae

To construct rough strains of Streptococcus pneumoniae in which the capsule locus was completely deleted, a genetic cassette to be used as a donor DNA in transformation was developed. The cassette contained an aphIII gene, conferring kanamycin resistance, flanked by segments of dexB and aliA. Since, in all strains of S. pneumoniae the capsule locus is between dexB and aliA, the DNA segments of these two genes allow insertion of a 1354-bp DNA fragment containing aphIII into the pneumococcal chromosome, determining the deletion of the whole capsule locus. The capsule locus was deleted from the classic type 2 and type 3 Averys strains, from R6 (whose complete genome sequence is released) and Rx1 (the two most commonly used transformation recipients), from a type 3 clinical strain and type 19F clinical isolate G54 (whose draft genome sequence is annotated). The effect of capsule removal was tested in 4 isogenic pairs. In unencapsulated strains, growth rate increased up to 56% and transformation frequency increased up to 1075-fold. A correlation was observed between the increase in growth rate and an increase in transformation frequency.

Tn2009, a Tn916-like element containing mef(E) in Streptococcus pneumoniae

ABSTRACT The association between the macrolide efflux gene mef(E) and the tet(M) gene was studied in two clinical strains of Streptococcus pneumoniae that belonged to serotypes 19F and 6A, respectively, and that were resistant to both tetracycline and erythromycin. The mef(E)-carrying element mega (macrolide efflux genetic assembly; 5,511 bp) was found to be inserted into a Tn916-like genetic element present in the chromosomes of the two pneumococcal strains. In both strains, mega was integrated at the same site, an open reading frame identical to orf6 of Tn916. The new composite element, Tn2009, was about 23.5 kb and, with the exception of the tet(M)-coding sequence, appeared to be identical in both strains. By sequencing of the junction fragments of Tn2009 at the site of insertion into the chromosome, it was possible to show that (i) the insertion site was identical in the two clinical strains and (ii) the integration of Tn2009 caused a 9.5 kb-deletion in the pneumococcal chromosome. It was not possible to detect the conjugal transfer of Tn2009 to a recipient pneumococcal strain; however, transfer of the whole element by transformation was shown to occur. It is possible to hypothesize that Tn2009 relies on transformation for its spread among clinical strains of S. pneumoniae.