Proline-rich Repeat Region Research Articles

Epidemiological investigations and locally determined genotype diversity of Mycoplasma synoviae in Central China from 2017 to 2019

Mycoplasma synoviae (M. synoviae) has been identified worldwide to cause respiratory diseases, infectious synovitis, airsacculitis, and eggshell apex abnormalities (EAA) in commercial chickens, which results in substantial economic losses to the poultry industry. Therefore, in this study, 258 flocks were investigated between 2017 and 2019 for M. synoviae by screening samples from Central China. Subsequently, 129 M. synoviae strains were isolated, with a positive rate of 50%. Moreover, a higher incidence of M. Synoviae infections was in layers (74.1%) than in broilers (20%) in this study. The 5′-end conserved segment of the variable lipoprotein hemagglutinin A (vlhA) gene of these isolates was then cloned and sequenced because it is a common genomic target identified so far for M. synoviae genotyping. Genotyping of all isolates was based on the phylogenetic analysis and length analysis of the proline-rich-repeat (PRR) regions, respectively. Phylogenetic analysis based on 5′-end conserved segment of the vlhA gene (76–421 nt) assigned the majority of the occurring strains as being from group 6, and others from groups 2 and 3. Results identified that these isolates were of 6 types: A (38aa), D (23aa), E (19aa), I (28aa), J (20aa), and L (35aa), based on the size of the PRR region analysis. Furthermore, most of the isolates (81.4% were identified as type L. Additionally, the epidemic types included only I and L in 2017; however, the types rose to 5 (A, D, E, I, L) in 2018 and rose to 6 (A, D, E, I, J, L) in 2019. These data showed the genotype diversity of M. synoviae in Central China. The high rate of positive flocks suggests the urgent need to take real-time supervisory controls of this Mycoplasma species in avian flocks.

Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS.

Members of the conserved ubiquilin (UBQLN) family of ubiquitin (Ub) chaperones harbor an antipodal UBL (Ub-like)-UBA (Ub-associated) domain arrangement and participate in proteasome and autophagosome-mediated protein degradation. Mutations in a proline-rich-repeat region (PRR) of UBQLN2 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD); however, neither the normal functions of the PRR nor impacts of ALS-associated mutations within it are well understood. In this study, we show that ALS mutations perturb UBQLN2 solubility and folding in a mutation-specific manner. Biochemical impacts of ALS mutations were additive, transferable to UBQLN1, and resulted in enhanced Ub association. A Drosophila melanogaster model for UBQLN2-associated ALS revealed that both wild-type and ALS-mutant UBQLN2 alleles disrupted Ub homeostasis; however, UBQLN2ALS mutants exhibited age-dependent aggregation and caused toxicity phenotypes beyond those seen for wild-type UBQLN2. Although UBQLN2 toxicity was not correlated with aggregation in the compound eye, aggregation-prone UBQLN2 mutants elicited climbing defects and neuromuscular junctions (NMJ) abnormalities when expressed in neurons. An UBA domain mutation that abolished Ub binding also diminished UBQLN2 toxicity, implicating Ub binding in the underlying pathomechanism. We propose that ALS-associated mutations in UBQLN2 disrupt folding and that both aggregated species and soluble oligomers instigate neuron autonomous toxicity through interference with Ub homeostasis.

Differences in virulence repertoire and cell invasive potential of Group A Streptococcus emm1-2 in comparison to emm1 genotype

Group A streptococcus (GAS, Streptococcus pyogenes) type emm1 is widely associated with streptococcal invasive disease. This type is prevalent worldwide but is rare in India. Instead, emm1-2 type which is closely related to emm1 but is a distinct type is more prevalent. Although emm1 has been well characterized, information available on emm1-2 is rare. In this study we present a comparative study of both types. DNA microarray analysis showed segregation of emm1 and emm1-2 isolates into two distinct clusters. Out of 229 arrayed genes, 83–87% were present, 6–9% absent and 4–8% genes were ambiguous in emm1 isolates. emm1-2 strains harboured only 68–77%, 11–13% were absent and 10–20% ambiguous genes. Fourteen genes, present in all emm1, were completely absent in the emm1-2 isolates. sfb1 is a gene which encodes for Streptococcal fibronectin binding adhesin and invasin which has restricted distribution among different emm types of GAS. A variant of sfb1 (sfb1-2) was the only gene which was present in all emm1-2 isolates, but absent from all emm1 strains. Sfb1 and Sfb1-2 differ in sequences in the aromatic domain and the proline rich repeat region, whereas the fibronectin binding region was conserved and exhibited similar fibronectin binding activity. The presence of Sfb1-2 in emm1-2 strains was concomitant with significantly higher fibronectin-binding and invasion efficiency of HEp-2 cells when compared to emm1 isolates. The role of Sfb1-2 in invasion was confirmed by latex bead assay. emm1-2 isolates follow membrane ruffling mechanism during invasion and intracellularly follow classical endocytic pathway. Further studies are required to understand the correlation between the presence of emm1-2 isolates and the disease pattern in North India.

Epstein-Barr Virus Latent Membrane Protein 1 Genetic Variability in Peripheral Blood B Cells and Oropharyngeal Fluids

We report the diversity of latent membrane protein 1 (LMP1) gene founder sequences and the level of Epstein-Barr virus (EBV) genome variability over time and across anatomic compartments by using virus genomes amplified directly from oropharyngeal wash specimens and peripheral blood B cells during acute infection and convalescence. The intrahost nucleotide variability of the founder virus was 0.02% across the region sequences, and diversity increased significantly over time in the oropharyngeal compartment (P = 0.004). The LMP1 region showing the greatest level of variability in both compartments, and over time, was concentrated within the functional carboxyl-terminal activating regions 2 and 3 (CTAR2 and CTAR3). Interestingly, a deletion in a proline-rich repeat region (amino acids 274 to 289) of EBV commonly reported in EBV sequenced from cancer specimens was not observed in acute infectious mononucleosis (AIM) patients. Taken together, these data highlight the diversity in circulating EBV genomes and its potential importance in disease pathogenesis and vaccine design. This study is among the first to leverage an improved high-throughput deep-sequencing methodology to investigate directly from patient samples the degree of diversity in Epstein-Barr virus (EBV) populations and the extent to which viral genome diversity develops over time in the infected host. Significant variability of circulating EBV latent membrane protein 1 (LMP1) gene sequences was observed between cellular and oral wash samples, and this variability increased over time in oral wash samples. The significance of EBV genetic diversity in transmission and disease pathogenesis are discussed.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Molecular interactions of alanine‐rich and proline‐rich regions of cell surface protein antigen c in Streptococcus mutans

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans, and its cell surface protein antigen c (PAc) is known to be associated with sucrose-independent adhesion to tooth surfaces. PAc is composed of several domains, including an N-terminal signal sequence, an alanine-rich repeat region (A-region), a proline-rich repeat region (P-region), and an anchor region. To investigate the functions of each domain, an A-region-deficient mutant strain of S. mutans was constructed, and recombinant PAc and A- and P-region proteins were also constructed. The interactions of each domain with the recombinant proteins were analyzed using surface plasmon resonance spectroscopy with a biomolecular interaction analyzing system. The A-region-deficient mutant strain showed the lowest levels of adherence to saliva-coated hydroxyapatite. Furthermore, findings in an immunoblot assay indicated that the A-region protein reacted strongly with proline-rich proteins in saliva, while the recombinant P-region protein interacted more quickly with PAc than the recombinant A-region protein. These results suggest that the A-region has a strong relationship with adhesion to tooth surfaces, while the P-region has a high affinity for PAc.

Miniature protein ligands for EVH1 domains: interplay between affinity, specificity, and cell motility.

Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins-Mena, VASP, and Evl-are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. It has previously been reported that a novel miniature protein, pGolemi, binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven Listeria monocytogenes motility. Here, scanning mutagenesis was used to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation.

Genetic diversity of the C protein β-antigen gene and its upstream regions within clonally related groups of type Ia and Ib group B streptococci

C protein beta antigen (Bac), a surface protein of group B streptococci (GBS), is known to concurrently bind the Fc portion of IgA and factor H (FH). The authors' previous work has demonstrated that mRNA expression levels show diversity among clonally related strains containing genes (bac) encoding Bac, with high expression noted in invasive strains. In this study, the bac gene and upstream regions containing putative promoters, three ORFs and an IS1381 insertion sequence were characterized. Three invasive strains showed high bac expression levels and did not show any notable mutations except one strain producing Bac that was able to bind FH but not IgA. A deletion of 51 amino acid residues, including part of the Bac IgA-binding region, was identified and hypothesized to contribute to the loss of the IgA-binding ability of this strain. A vaginal strain that showed somewhat higher bac expression levels and produced Bac lacking immunoreactivity contained an 11 bp deletion, which generated a premature termination codon, in the region preceding the IgA-binding region. In another vaginal strain that did not express bac, disruption of the upstream ORFs of the sensor histidine kinase and DNA-binding response regulator, due to frameshift mutations, was noted although it is not known whether these proteins directly affect bac expression levels. An IS1381 insertion into the promoter region was found in another vaginal strain that showed low expression levels and produced Bac with a significantly larger proline-rich repeat region. These results demonstrate considerable genetic diversity of the bac and upstream regions of invasive and noninvasive GBS, which may contribute to the variability of bac expression levels among those strains.

Paired-like homeoprotein ESXR1 acts as a sequence-specific transcriptional repressor of the human K-ras gene

Gain-of-function mutation of the K-ras gene is one of the most common genetic changes in human tumors. In tumors carrying K-ras mutation, the presence of oncogenic K-Ras is necessary for maintenance of the transformed phenotype. ESXR1 is a human paired-like homeodomain-containing protein expressed primarily in the testis. In cells, the 65-kDa full-length ESXR1 protein is proteolytically processed into an N-terminal 45-kDa fragment containing the homeodomain, which localizes exclusively within the nucleus, and a C-terminal 20-kDa fragment consisting of a proline-rich repeat region, which is located in the cytoplasm. In this work, we demonstrated that the N-terminal ESXR1 fragment specifically recognizes the TAATNNNATTA P3 consensus sequence for the paired-like homeodomain and functions as a sequence-specific transcriptional repressor. We also showed that the N-terminal ESXR1 fragment binds to the TAATGTTATTA sequence present within the first intron of the human K-ras gene and inhibits its expression at both mRNA and protein levels. Ectopic expression of the N-terminal ESXR1 fragment in human carcinoma cells that carry mutated K-ras reduces the level of K-Ras and thereby inhibits the tumor cell proliferation. Identification of ESXR1 as a transcriptional repressor of K-ras has an important implication for the development of cancer therapy that inhibits oncogenic K-Ras expression.

The Mycobacterial Heparin-Binding Hemagglutinin Is a Protective Antigen in the Mouse Aerosol Challenge Model of Tuberculosis

The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a approximately 0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.

Contribution of the alanine-rich region of Streptococcus mutans P1 to antigenicity, surface expression, and interaction with the proline-rich repeat domain.

Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli, and (v) in E. coli, P1 is secreted in the absence of SecB.

Paired-like homeodomain protein ESXR1 possesses a cleavable C-terminal region that inhibits cyclin degradation.

The eukaryotic cell cycle is regulated by sequential activation and inactivation of cyclin-cyclin-dependent kinase (Cdk) complexes. In this work, we screened human cDNAs that can rescue yeast Saccharomyces cerevisiae from lethality caused by ectopic expression of human cyclin E and isolated a cDNA encoding ESXR1, a paired-like homeodomain-containing protein with a unique C-terminal proline-rich repeat region. In adult tissues, ESXR1 is primarily expressed in the testis. We demonstrate that ESXR1 prevents degradation of ubiquitinated cyclins in human cells. Accordingly, elevation of ESXR1 level results in accumulation of cyclin A and cyclin B1 and thereby provokes M-phase arrest. In human cells, the 65-kDa full-length ESXR1 protein is capable of proteolytically processing into N-terminal 45-kDa and C-terminal 20-kDa fragments. The C-terminal fragment, containing a proline-rich repeat region, is localized to the cytoplasm and displays the ability to inhibit cyclin degradation. In contrast, the N-terminal fragment, containing a paired-like homeodomain, is localized exclusively in the nucleus, suggesting that it plays a role in transcription. Our results indicate that proteolytic processing of ESXR1 plays a role in concerted regulation of the cell cycle and transcription in human cells.

Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines.

The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.

LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes.

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogenes EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes.

Characterization of domains in mice of calnexin-t, a putative molecular chaperone required in sperm fertility, with use of glutathione S-transferase-fusion proteins.

Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.

The ActA polypeptides of Listeria ivanovii and Listeria monocytogenes harbor related binding sites for host microfilament proteins

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes acts as a nucleator protein, generating the actin cytoskeleton around intracellularly motile bacteria. In this work, we examined the functional similarity of ActA from Listeria ivanovii (iActA) ATCC 19119 to its L. monocytogenes counterpart. The amino acid sequence of iActA predicts a molecular mass of 123 kDa and harbors eight proline-rich repeats. For functional analysis, various iActA derivatives and hybrid constructs of L. ivanovii and L. monocytogenes ActA polypeptides were transiently expressed in epithelial cells and examined for recruitment of host microfilament proteins by a mitochondrial targeting assay. As has been demonstrated with ActA, iActA also spontaneously inserted into the surface of mitochondria and induced recruitment of actin, alpha-actinin, and the vasodilator-stimulated phosphoprotein (VASP) to these subcellular organelles. By comparison of amino-terminally truncated iActA derivatives for their ability to recruit cytoskeletal proteins, a region essential for actin filament accumulation was identified between amino acid residues 290 and 325. Such derivatives, however, retained their ability to bind VASP. Replacement of the proline-rich repeats in ActA with those of iActA also resulted in VASP recruitment. Hence, despite the limited overall sequence homology between ActA and iActA, the two molecules consist of at least two similar domains: a highly positively charged N-terminal domain that is directly involved in actin filament recruitment and a proline-rich repeat region required for VASP binding.

The actin-polymerization protein from Listeria ivanovii is a large repeat protein which shows only limited amino acid sequence homology to actA from Listeria monocytogenes

Within infected eukaryotic cells the two pathogenic Listeria species, L. monocytogenes and L. ivanovii, induce polymerization of cellular actin and the formation of a propulsive actin tail at one bacterial pole. For L. monocytogenes it has been shown that the product of the listerial actA gene is required for this process which is regarded as a model for actin-based motility. We have now cloned and sequenced a functionally analogous gene from L. ivanovii; its product, as deduced from the DNA sequence, is considerably larger (108 kDa) than L. monocytogenes ActA (67 kDa) and shares only a limited amino acid sequence homology (46% similarity on average) with the latter protein. This is the first example of a virulence gene product from L. ivanovii which is significantly different from its L. monocytogenes counterpart. Comparison of the two ActA proteins gives new insight into the structure of this class of actin-polymerization proteins, in particular with respect to their proline-rich repeat region.

The actin-polymerization protein from Listeria ivanovii is a large repeat protein which shows only limited amino acid sequence homology to ActA from Listeria monocytogenes.

Within infected eukaryotic cells the two pathogenic Listeria species, L. monocytogenes and L. ivanovii, induce polymerization of cellular actin and the formation of a propulsive actin tail at one bacterial pole. For L. monocytogenes it has been shown that the product of the listerial actA gene is required for this process which is regarded as a model for actin-based motility. We have now cloned and sequenced a functionally analogous gene from L. ivanovii; its product, as deduced from the DNA sequence, is considerably larger (108 kDa) than L. monocytogenes ActA (67 kDa) and shares only a limited amino acid sequence homology (46% similarity on average) with the latter protein. This is the first example of a virulence gene product from L. ivanovii which is significantly different from its L. monocytogenes counterpart. Comparison of the two ActA proteins gives new insight into the structure of this class of actin-polymerization proteins, in particular with respect to their proline-rich repeat region.

Affinity and specificity of the interactions between Streptococcus mutans antigen I/II and salivary components.

Adherence to salivary pellicle-coated tooth surfaces and aggregation by salivary components of Streptococcus mutans involves a major cell surface protein termed antigen (Ag) I/II. The objectives of this study were to evaluate the affinity and specificity of the interactions between AgI/II and human saliva in assays of 125I-AgI/II binding to saliva-coated hydroxyapatite (SHA) and of S. mutans aggregation by salivary agglutinin (SAG), monitored turbidimetrically. 125I-AgI/II binding to SHA followed saturation kinetics, and Scatchard plot analysis indicated two binding sites with dissociation constants of the order of 10(-10) mol/L and 10(-9) mol/L. The binding to SHA of the C-terminal one-third of AgI/II which corresponds to AgII was less than one-fifth that of the whole molecule and did not show evidence of saturation. The binding of 125I-AgI/II was inhibited by native or recombinant fragments that mapped in the N-terminal part of the molecule and that contained the alanine-rich repeat region, whereas fragments mapping at the central or C-terminal one-third had no effect. As with binding to SHA, the regions of AgI/II which inhibited aggregation mapped at the N-terminal part of the molecule, but, in addition, a recombinant segment mapping at the central part and containing the proline-rich repeat region was also inhibitory. The S. mutans-aggregating activity of SAG or whole saliva was inhibited by amino compounds, and most strongly by L-lysine and analogues possessing omega-primary amine groups. These data support the role of AgI/II as an adhesin with high-affinity binding for SHA receptors, mediated by the N-terminal part of the molecule. This region is also involved in SAG-induced S. mutans aggregation, which is sensitive to amino compounds.

The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.

Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.