Identification and Functional Analysis of the S-Layer Protein SplA of Paenibacillus larvae, the Causative Agent of American Foulbrood of Honey Bees

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.     

C3larvin Toxin,an ADP-ribosyltransferase from Paenibacillus larvae

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD⁺ (K_m = 34 ± 12 μm) and RhoA (K_m = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (K_i = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.     

The N-terminus of Paenibacillus larvae C3larvinA modulates catalytic efficiency

C3larvinA was recently described as a mono-ADP-ribosyltransferase (mART) toxin from the enterobacterial repetitive intergenic consensus (ERIC) III genotype of the agricultural pathogen, Paenibacillus larvae. It was shown to be the full-length, functional version of the previously described C3larvin_trunc toxin, due to a 33-residue extension of the N-terminus of the protein. In the present study, a series of deletions and substitutions were made to the N-terminus of C3larvinA to assess the contribution of the α₁-helix to toxin structure and function. Catalytic characterization of these variants identified Asp²³ and Ala³¹ residues as supportive to enzymatic function. A third residue, Lys³⁶, was also found to contribute to the catalytic activity of the enzyme. Analysis of the C3larvinA homology model revealed that these three residues were participating in a series of interactions to properly orient both the Q-X-E and S-T-S motifs. Ala³¹ and Lys³⁶ were found to associate with a structural network of residues previously identified in silico, whereas Asp²³ forms novel interactions not previously described. At last, the membrane translocation activity into host target cells of each variant was assessed, highlighting a possible relationship between protein dipole and target cell entry.     

Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees

Aims: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. Methods and Results: For this purpose, we established a protocol for the transformation of the plasmid pAD43‐25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re‐germination. Conclusions: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP‐tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re‐germination. Significance and Impact of the Study: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.     

Negative Correlation between Individual-Insect-Level Virulence and Colony-Level Virulence of Paenibacillus larvae,the Etiological Agent of American Foulbrood of Honeybees

Paenibacillus larvae is the etiological agent of American foulbrood (AFB) in honeybees. Recently, different genotypes of P. larvae (ERIC I to ERIC IV) were defined, and it was shown that these genotypes differ inter alia in their virulence on the larval level. On the colony level, bees mitigate AFB through the hygienic behavior of nurse bees. Therefore, we investigated how the hygienic behavior shapes P. larvae virulence on the colony level. Our results indicate that P. larvae virulence on the larval level and that on the colony level are negatively correlated.American foulbrood (AFB) is among the economically most important honeybee diseases. The etiological agent of AFB is the gram-positive, spore-forming bacterium Paenibacillus larvae (9). The extremely tenacious spores are the infectious form of this organism. These spores drive disease transmission within colonies (11), as well as between colonies as soon as they end up in the honey stores of an infected colony (12).The species P. larvae can be subdivided into four different genotypes designated ERIC I to ERIC IV based on results from repetitive-element PCR (20) using enterobacterial repetitive intergenic consensus (ERIC) primers (9, 10), with P. larvae ERIC I and ERIC II being the two practically most important genotypes (1, 2, 9, 10, 13, 16). The four genotypes were shown previously to differ in phenotype, including virulence on the larval level (8, 9). While larvae infected with genotypes ERIC II to ERIC IV were killed within only 6 to 7 days, it took P. larvae ERIC I around 12 to 14 days to kill all infected individuals. Therefore, genotype ERIC I was considered to be less virulent and the other three genotypes were considered to be highly virulent (7-9) on the larval level.P. larvae is an obligately killing pathogen which must kill its host to be transmitted. The virulence of such an obligate killer is thought to be determined primarily by two factors, (i) the probability of infecting a host and (ii) the time to host death (6). The problem of ensuring a high enough probability of infecting the next host is solved for P. larvae by (i) the tenacious exospores, which remain infectious for over half a century (17) and, therefore, can wait for decades for the next host to pass by, and (ii) a high pathogen reproduction rate (23) and, thus, the production of an extremely high number of spores within each infected larva.For evaluating the second factor determining P. larvae virulence, the time to host death, it is important to consider the two levels of honeybee hosts, the level of the individual larva dying from AFB and the level of the colony succumbing to AFB.The virulence of P. larvae genotypes on the larval level has been analyzed recently (8, 9). We have now determined the colony-level virulence for the two most common and practically important (10, 16) genotypes of P. larvae, ERIC I and ERIC II, significantly differing in virulence on the larval level (8). We will discuss how the time to larval death relates to the time to colony death and how the hygienic response shapes P. larvae virulence.     

Unbiased random mutagenesis contributes to a better understanding of the virulent behaviour of Paenibacillus larvae

Aims

American foulbrood, caused by the Gram‐positive bacteria Paenibacillus larvae, is one of the most severe bacterial diseases of the European honey bee. The bacterium has been known for long, but only the last decade the mechanisms used by the pathogen to cause disease in its host are starting to unravel. In this study, the knowledge of this virulent behaviour is expanded and several possible virulence factors are suggested.

Methods and Results

Identification of possible virulence factors has been done by random mutagenesis to ensure an unbiased approach. A library of mutants was tested for a significant difference in virulence using in vitro exposure assays. Affected loci were characterized and their potential to contribute in virulence of the pathogen was assessed.

Conclusions

The identified mutated loci dacB, dnaK, metN, ywqD, lysC, serC and gbpA are known to encode for virulence factors in other bacteria and are suggested to play a similar role in P. larvae.

Significance and Impact of the Study

The study identified new possible virulence factors for P. larvae genotype ERIC I in an unbiased way. This contributes to the knowledge and understanding of the possible mechanisms used by this pathogen to colonize and kill its host.     

Multiple Locus Variable number of tandem repeat Analysis: A molecular genotyping tool for Paenibacillus larvae

American Foulbrood, caused by Paenibacillus larvae, is the most severe bacterial disease of honey bees (Apis mellifera). To perform genotyping of P. larvae in an epidemiological context, there is a need of a fast and cheap method with a high resolution. Here, we propose Multiple Locus Variable number of tandem repeat Analysis (MLVA). MLVA has been used for typing a collection of 209 P. larvae strains from which 23 different MLVA types could be identified. Moreover, the developed methodology not only permits the identification of the four Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes, but allows also a discriminatory subdivision of the most dominant ERIC type I and ERIC type II genotypes. A biogeographical study has been conducted showing a significant correlation between MLVA genotype and the geographical region where it was isolated.     

Structural variability of C3larvin toxin. Intrinsic dynamics of the α/β fold of the C3-like group of mono-ADP-ribosyltransferase toxins

C3larvin toxin is a new member of the C3 class of the mono-ADP-ribosyltransferase toxin family. The C3 toxins are known to covalently modify small G-proteins, e.g. RhoA, impairing their function, and serving as virulence factors for an offending pathogen. A full-length X-ray structure of C3larvin (2.3 Å) revealed that the characteristic mixed α/β fold consists of a central β-core flanked by two helical regions. Topologically, the protein can be separated into N and C lobes, each formed by a β-sheet and an α-motif, and connected by exposed loops involved in the recognition, binding, and catalysis of the toxin/enzyme, i.e. the ADP-ribosylation turn–turn and phosphate–nicotinamide PN loops. Herein, we provide two new C3larvin X-ray structures and present a systematic study of the toxin dynamics by first analyzing the experimental variability of the X-ray data-set followed by contrasting those results with theoretical predictions based on Elastic Network Models (GNM and ANM). We identify residues that participate in the stability of the N-lobe, putative hinges at loop residues, and energy-favored deformation vectors compatible with conformational changes of the key loops and 3D-subdomains (N/C-lobes), among the X-ray structures. We analyze a larger ensemble of known C3bot1 conformations and conclude that the characteristic ‘crab-claw’ movement may be driven by the main intrinsic modes of motion. Finally, via computational simulations, we identify harmonic and anharmonic fluctuations that might define the C3larvin ‘native state.’ Implications for docking protocols are derived.     

The novel type II toxin–antitoxin PacTA modulates Pseudomonas aeruginosa iron homeostasis by obstructing the DNA-binding activity of Fur

Type II toxin–antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes and are involved in diverse critical cellular functions such as defense against phages, biofilm formation, persistence, and virulence. GCN5-related N-acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. We here describe a group of GNAT-Xre TA modules widely distributed among Pseudomonas species. We investigated PacTA (one of its members encoded by PA3270/PA3269) from Pseudomonas aeruginosa and demonstrated that the PacT toxin positively regulates iron acquisition in P. aeruginosa. Notably, other than arresting translation through acetylating aminoacyl-tRNAs, PacT can directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit the expression of downstream iron-acquisition-related genes. We further showed that the expression of the pacTA locus is upregulated in response to iron starvation and the absence of PacT causes biofilm formation defect, thereby attenuating pathogenesis. Overall, these findings reveal a novel regulatory mechanism of GNAT toxin that controls iron-uptake-related genes and contributes to bacterial virulence.     

Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

Background

Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.

Methodology/Principal Findings

An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.

Conclusions/Significance

The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.     

Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.     

Synergistic effect of surfactin from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C4 and <Emphasis Type="Italic">Achyrocline satureioides</Emphasis> extracts on the viability of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml⁻¹ and the HE concentration from 32 to 4 μg ml⁻¹, values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.     

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

Background

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.

Results

The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.

Conclusion

Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.     

Localization of Putative Virulence Genes on a Physical Map of the Bacillus thuringiensis subsp. gelechiae Chromosome

The insect pathogen Bacillus thuringiensis (Bt) has earlier been shown to possess virulence factors in addition to the crystal toxins. Bt subsp. gelechiae strain Bt13 lacks crystals but is still virulent to lepidopteran insects. Among the virulence co-expressed genes are two phospholipases; phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-degrading phospholipase C (PC-PLC), flagellin, and β-lactamase I. In addition to these putative virulence factors the toxic neutral metalloprotease immune inhibitor A (InA) has been identified. In this paper we report a circular 5.9 Mb combined physical and genetic map of the of the Bt subsp. gelechiae chromosome. The genes encoding PI-PLC, PC-PLC, InA, flagellin, and β-lactamase I are shown to be scattered over the chromosome. The PLC-encoding genes have been cloned from Bt13, and DNA sequencing showed that the Bt subsp. gelechiae PLC genes are >90% identical to their previously cloned equivalents from Bt or B. cereus. An HD-1 crystal toxin (cryIA) gene probe was found to hybridize to the Bt13 chromosome, but not to extrachromosomal elements. Received: 26 March 1998 / Accepted: 6 May 1998     

ERIC-PCR Genotyping of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> in Southern Italian Honey and Brood Combs

Given the considerable economic loss to beekeepers worldwide and the possible public health implications related to the presence of antibiotics in honey, an American Foulbrood (AFB) monitoring/prevention program for Paenibacillus larvae is regarded as essential. This study investigates the occurrence and distribution of P. larvae genotypes in honey and brood combs from Apulia (Italy). Genotyping of P. larvae isolates using ERIC-PCR generated a total of four different ERIC banding patterns (ERIC-A, ERIC-B, ERIC-C, ERIC-D), including fragments ranging from 200 to 3000 bp. Considering that the genotype has an influence on P. larvae infections and multi-genotype infections of colonies or apiaries may increase the complexity of P. larvae infections by influencing the type and speed of the development of clinical symptoms, the findings of the present study could be helpful for training veterinarians, bee inspector’s extension staff, and beekeepers, thus improving the detection of AFB infections in the field.     

Molecular Typing of Paenibacillus larvae Strains Isolated from Bulgarian Apiaries Based on Repetitive Element Polymerase Chain Reaction (Rep-PCR)

The aim of the present study was to perform molecular typing of Paenibacillus larvae (P. larvae) isolates from Bulgarian apiaries with repetitive element polymerase chain reaction (rep-PCR) using BOX A1R, MBO REP1, and ERIC primers. A total of 96 isolates collected from brood combs with clinical symptoms of American foulbrood originating from apiaries located in different geographical regions of Bulgaria, a reference strain P. larvae NBIMCC 8478 and 30 commercial honey samples with Bulgarian origin were included in the study. Rep-PCR fingerprinting analysis revealed two genotypes ab and AB of P. larvae isolates from brood combs and honey samples. A combination of genotypes ab/AB was detected in one apiary and honey sample. The prevailing genotype ab was found in 78.1 % of brood combs isolates as well as in the reference strain whereas genotype AB was determined in 21.9 % of isolates. The examination of honey samples confirmed the preponderance of ab genotype which was demonstrated in 20 of 30 samples analyzed. In conclusion, the genetic epidemiology of P. larvae revealed two genotypes—ab and AB for Bulgarian strains. Developed protocols for molecular typing of P. larvae are reliable and may be used to trace the source of infection.     

Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.