Andreas Podbielski

Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS)

Ten novel streptococcal shuttle vectors for genomic integration and allelic replacements have been constructed based on plasmid pSF152. These vectors can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The basic vector pFW5 (2.8 kb, aad9 spectinomycin-resistance marker) carries two multiple cloning sites MCS-I and MCS-II (10 and 15 restrictions sites, respectively) to either side of the aad9 resistance gene. Each MCS is flanked by transcription termination sites for stabilization of recombinant plasmids. In vector pFW6 the transcription terminator between aad9 and MCS-II was deleted. Plasmids pFW7 through pFW10 carry resistance genes for kanamycin, chloramphenicol, erythromycin, and tetracyclin instead of aad9. Vectors pFW11 and pFW12 are pFW5/6 derivatives harboring an improved synthetic aad9 promoter. In pFW-phoA and pFW-gfp, promoterless alkaline phosphatase and green fluorescent protein boxes were integrated into MCS-I. If streptococcal DNA fragments are cloned into MCS-I and MSC-II, these vectors can be used for specific allelic replacements in streptococci via double-crossover recombinations. Depending on the vector used, this event will not lead to polar effects, facilitating mutagenesis within operons. The vectors containing reporter boxes allow in vivo studies of gene expression and promoter activity in pathogenic streptococci and potentially, also in other Gram-positive bacteria.

Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci

Evaluation of homologous regions of published M protein (emm) gene sequences from group A streptococci (GAS; Streptococcus pyogenes) was used to design three primer pairs for polymerase chain reaction (PCR) and three oligonucleotide probe sequences internal to the amplified products. One set of primers and corresponding probe should detect and lead to amplification of emm(-like) genes of virtually every type (“all M”), another (“SOR-M”) should only amplify emm(-like) genes from GAS negative for serum opacity reaction (SOR) and the third (“SOR+M”) should expand only emm(-like) genes from SOR+ GAS. Using the “all M” primer pair for PCR on the genomic DNA from GAS of 29 different M types as well as from a group C and a group G streptococcal isolate, DNA fragments within the expected size range were amplified in every assay. All PCR products reacted with the “all M” probe. Related sequences were not detected in genomic DNA of an S. agalactiae and an Enterococcus faecalis isolate. Applying the “SOR-M” and “SOR+M” primers to identical assays led to mutually exclusive amplification products. The “SOR+M” and “SOR+M” probes hybridized only to their corresponding products. Exceptions to this exclusivity were the SOR+ GAS of M types 3, 8, 27, 34, 42, 67, and 69, which consistently reacted only with the “SOR+M” primer/probe set. Analysis of sequence data from the amplified emm(-like) 2, 3, 18, and 19 genes revealed interesting specific features such as conserved gaps in the C-terminal sequence regions from SOR+ and the exceptional SOR- GAS strains. These data indicate the existence of a subgroup of strains among SOR- GAS and may advance our understanding of phylogenetic relationship between different serotypes of GAS.

M‐related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono‐phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M‐related proteins, Mrp and Enn, which have been described as IgG‐ and IgA‐binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M‐untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony‐forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild‐type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM‐labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild‐type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis‐sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1u2003h of co‐incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro‐rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.

Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb.

Beta‐haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16u2003kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.

MOLECULAR CHARACTERIZATION OF GROUP A STREPTOCOCCAL (GAS) OLIGOPEPTIDE PERMEASE (OPP) AND ITS EFFECT ON CYSTEINE PROTEASE PRODUCTION

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 10u2003192u2003bp 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.

What is the size of the group A streptococcal vir regulon ? The Mga regulator affects expression of secreted and surface virulence factors

Abstract 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.

Surface protein‐CAT reporter fusions demonstrate differential gene expression in the wr regulon of Streptococcus pyogenes

Streptococcus pyogenes expresses at least two virulence factors, the anti‐phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)‐derived potential promoter regions of the virR, M protein (emm12), and ScpA (scpA) genes from an M type 12 5. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive trans‐acting regulator of emm12 and scpA expression; however, virR is not autoregulated. A potential virR‐bindlng consensus sequence is postulated for emm12, scpA and other M‐like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation.

Nucleotide substitutions and small‐scale insertion produce size and antigenic variation in group A streptococcal M1 protein

The presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type‐specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N‐terminal non‐repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N‐terminal non‐repeat domains of two other closely related strains, 71‐155 and 76‐088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N terminal non‐repeat domain of the M protein of GAS.

Three different types of organization of the vir regulon in group A streptococci

SummaryThe DNA of group A streptococci (GAS) encodes several important virulence factors such as the antiphagocytic M protein, the Ig-Fc-binding M-related proteins (FcrA-like and EnnX-like) and the complement factor-inactivating C5a peptidase. The corresponding genes emm, fcrA, ennX, and scpA, respectively, were assumed to be located close together in the GAS genome. Additionally, emm and scpA have been found to be under the positive, coordinate control of the virR locus, which led to the designation “vir regulon” for the corresponding genomic segment. In order to map the vir regulons of many GAS serotypes and to analyse any correlation between the organization of vir regulons and circumscribed heterogeneities within the emm, virR, and scpA genes, an approach using several distinct sets of polymerase chain reaction (PCR) experiments was chosen. By examination of the genomic DNA of 42 GAS isolates from 36 different M serotypes three patterns of vir regulon topography were found. The first, designated “large vir regulon” (LVR), consists of virR -fcrA(-like) emm - ennX(-like) - scpA. The second, designated “small vir regulon” (SVR), contains virR - emm- scpA, and the last, designated “unusual vir regulon” (UVR), resembles SVR but contains additional heterogeneous sequences between emm and scpA. The patterns correlate with heterogeneities at the 3′ ends of the virR and scpA genes, with the M classification system and the occurrence of specific non-coding intervening sequences within the vir regulons. The potential impact of these patterns on models to account for generation of vir regulons is discussed.

Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor

An internal fragment of the cfb gene from group B streptococcal (GBS) strain R268 was amplified by polymerase chain reaction (PCR) using degenerate primers with sequences derived from the CAMP-factor amino acid (aa) sequence of GBS strain NCTC8181 [Rühlmann et al. (1988) FEBS Lett 235: 262–266]. After cloning and sequencing this fragment, the remainder of cfb and the adjacent 5′ and 3′ sequences were amplified by inverted PCR of genomic DNA and directly sequenced from the PCR product. Within the 1560 bp sequenced, a complete cfb gene deviating in two deduced aa residues from the published sequence was identified. In addition, the cfbR268 sequence contained a 29-aa leader peptide. Using primers directed to the 5′ and 3′ ends of cfb for PCR, a cfb gene of uniform size could be detected in 19 clinical GBS isolates including three phenotypically CAMP-negative strains. Utilizing Northern blot analysis and primer extension assays, the cfbR268 promoter was located and the length of the cfb transcript was assessed at about 1100 bp. In a parallel experiment, no cfb transcript could be detected from the CAMP-negative GBS strain 74–360. The complete cfbR268 gene and different portions of its 5′ and 3′ ends were cloned into the plasmid pJLA602 and expressed in E. coli DH5α. The recombinant peptides could be detected by Western immunoblots with polyclonal antiserum. Only the full-sized recombinant CAMP-factor was found to exert co-hemolytic activity in a sheep-blood agar assay. This co-hemolytic activity could be inhibited by anti-CAMP antiserum.