Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism.

Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine-isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.

Induction of Necrosis in Human Macrophage Cell Lines by Corynebacterium diphtheriae and Corynebacterium ulcerans Strains Isolated from Fatal Cases of Systemic Infections.

When infecting a human host, Corynebacterium diphtheriae and Corynebacterium ulcerans are able to impair macrophage maturation and induce cell death. However, the underlying molecular mechanisms are not well understood. As a framework for this project, a combination of fluorescence microscopy, cytotoxicity assays, live cell imaging, and fluorescence-activated cell sorting was applied to understand the pathogenicity of two Corynebacterium strains isolated from fatal cases of systemic infections. The results showed a clear cytotoxic effect of the bacteria. The observed survival of the pathogens in macrophages and, subsequent, necrotic lysis of cells may be mechanisms explaining dissemination of C. diphtheriae and C. ulcerans to distant organs in the body.

Of mice and men: Interaction of Corynebacterium diphtheriae strains with murine and human phagocytes.

Seven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.

The C-terminal coiled-coil domain of Corynebacterium diphtheriae DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.

BACKGROUND: Corynebacterium diphtheriae is the etiologic agent of diphtheria and different systemic infections. The bacterium has been classically described as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of C. diphtheriae still remains unclear. Recently, the DIP0733 transmembrane protein was found to play an important role in the interaction with matrix proteins and cell surfaces, nematode colonization, cellular internalization and induction of cell death. RESULTS: In this study, we identified a number of short linear motifs and structural elements of DIP0733 with putative importance in virulence, using bioinformatic approaches. A C-terminal coiled-coil region of the protein was considered particularly important, since it was found only in DIP0733 homologs in pathogenic Corynebacterium species but not in non-pathogenic corynebacteria. Infections of epithelial cells and transepithelial resistance assays revealed that bacteria expressing the truncated form of C. diphtheriae DIP0733 and C. glutamicum DIP0733 homolog are less virulent, while the fusion of the coiled-coil sequence to the DIP0733 homolog from C. glutamicum resulted in increased pathogenicity. These results were supported by nematode killing assays and experiments using wax moth larvae as invertebrate model systems. CONCLUSIONS: Our data indicate that the coil-coiled domain of DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.

Anti-polysaccharide and anti-diphtheria protective antibodies after 13-valent pneumococcal conjugate vaccination in rheumatoid arthritis patients under immunosuppressive therapy.

Immunogenicity of 13-valent pneumococcal polysaccharide (PnPS) conjugate vaccine (PCV13) was evaluated in 38 rheumatoid arthritis patients under immunosuppressive treatment and 20 healthy controls (HC). Antibodies to all PnPS and diphtheria-toxin analogue conjugate protein were measured pre- (T0), 1 (T1), 6 (T2), 12 (T3) months post-immunization. Patients and HC had similar response to individual PnPS. Mean antibody levels to all PnPS but one doubled at T1 compared with T0, with T3 persistence for only 8-7/13 PnPS. Baseline antibody levels was inversely associated with the rate of responders at T1 (T1/T0≥2) to 11/13 PnPS. Few subjects reached protective IgG levels against some serotypes frequently isolated in Italian patients with invasive pneumococcal disease. Antibody response was not influenced by therapy, except the one to PS7F, which was reduced by tumor necrosis factor-α-inhibitors. Vaccination increased also anti-diphtheria IgG. Despite this study substantially confirmed the PCV13 immunogenicity in immunocompromised patients, it also revealed some limitations.

Microbe Profile: Corynebacterium diphtheriae - an old foe always ready to seize opportunity.

Corynebacterium diphtheriae is a globally important Gram-positive aerobic Actinobacterium capable of causing the toxin-mediated disease, diphtheria. Diphtheria was a major cause of childhood mortality prior to the introduction of the toxoid vaccine, yet it is capable of rapid resurgence following the breakdown of healthcare provision, vaccination or displacement of people. The mechanism and treatment of toxin-mediated disease is well understood, however there are key gaps in our knowledge on the basic biology of C. diphtheriae particularly relating to host colonisation, the nature of asymptomatic carriage, population genomics and host adaptation.

Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s.

BACKGROUND: Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 - 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains. RESULTS: C. diphtheriae strains from Belarus belong to ten sequence types (STs). Two major clones, non-toxigenic ST5 and toxigenic ST8, encompassed 76% of the isolates that are associated with sore throat and diphtheria in patients, respectively. Core genomic diversity is limited within outbreak-associated ST8 with relatively higher mutation rates (8.9 × 10-7 substitutions per strain per year) than ST5 (5.6 × 10-7 substitutions per strain per year) where most of the diversity was introduced by recombination. A variation in the virulence gene repertoire including the presence of tox gene is likely responsible for pathogenic differences between different strains. However, strains with similar virulence potential can cause disease in some individuals and remain asymptomatic in others. Eight synonymous single nucleotide polymorphisms were observed between the tox genes of the vaccine strain PW8 and other toxigenic strains of ST8, ST25, ST28, ST41 and non-toxigenic tox gene-bearing (NTTB) ST40 strains. A single nucleotide deletion at position 52 in the tox gene resulted in the frameshift in ST40 isolates, converting them into NTTB strains. CONCLUSIONS: Non-toxigenic C. diphtheriae ST5 and toxigenic ST8 strains have been endemic in Belarus both during and after the epidemic in 1990s. A high vaccine coverage has effectively controlled diphtheria in Belarus; however, non-toxigenic strains continue to circulate in the population. Recombination is an important evolutionary force in shaping the genomic diversity in C. diphtheriae. However, the relative role of recombination and mutations in diversification varies between different clones.

An isolated outbreak of diphtheria in South Africa, 2015.

An outbreak of respiratory diphtheria occurred in two health districts in the province of KwaZulu-Natal in South Africa in 2015. A multidisciplinary outbreak response team was involved in the investigation and management of the outbreak. Fifteen cases of diphtheria were identified, with ages ranging from 4 to 41 years. Of the 12 cases that were under the age of 18 years, 9 (75%) were not fully immunized for diphtheria. The case fatality was 27%. Ninety-three household contacts, 981 school or work contacts and 595 healthcare worker contacts were identified and given prophylaxis against Corynebacterium diphtheriae infection. A targeted vaccination campaign for children aged 6-15 years was carried out at schools in the two districts. The outbreak highlighted the need to improve diphtheria vaccination coverage in the province and to investigate the feasibility of offering diphtheria vaccines to healthcare workers.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

[The sensitivity to antibiotics of biofilm cultures of toxigenic strains Corynebacterium diphtheriae].

The article presents analysis of sensitivity to antibacterial preparations of typical and biofilm culture of museum strain of Corynebacterium diphtheriae gravis tox+ SV-665. The strain was obtained from the L.A. Tarasevitch state research institute of standardization and control of medical biological preparations. The second strain C. diphtheriaecirculates gravis tox+ circulates in population of the Rostov oblast and it was recovered from patient with diagnosis of "localized form of diphtheria" by bacteriologic laboratory "1002 CGSEN SKVO" of Rostov-on-Don. The week and month biofilm cultures of both strains of C. diphtheriae gravis tox+ were used. The sensitivity to antibacterial preparations of typical and biofilm cultures of museum and circulating in population strains of agent of diphtheria were detected using minimal suppressing concentration by technique of serial dilutions in fluid growth medium. It is demonstrated that the most effective in respect of C. diphtheriae are such preparations as cefotaxinum, gentamycinum, lincomycin, canamycin and cefasolin. The sensitivity of pathogen in composition of biofilm to these preparations has no changes.

The Trojan Horse of the microbiological arms race: phage-encoded toxins as a defence against eukaryotic predators.

Phage-encoded Shiga toxin (Stx) acts as a bacterial defence against the eukaryotic predator Tetrahymena. To function as an effective bacterial anti-predator defence, Stx must kill a broad spectrum of predators. Consistent with that assertion, we show here that bacterially encoded Stx efficiently kills the bacteriovore Acanthamoeba castellanii in co-culture. We also show that, in addition to Stx, the phage-encoded exotoxin, diphtheria toxin (Dtx) expressed by Corynebacterium diphtheriae also can function as part of an anti-predator strategy; it kills Acanthamoeba in co-culture. Interestingly, only exotoxins produced by bacteria internalized by the Acanthamoeba predator are cytolethal; the presence of purified Dtx or Stx in culture medium has no effect on predator viability. This finding is consistent with our results indicating that intoxication of Acanthamoeba by these exotoxins does not require a receptor. Thus bacteria, in the disguise of a food source, function as a 'Trojan Horse', carrying genes encoding an exotoxin into target organisms. This 'Trojan Horse' mechanism of exotoxin delivery into predator cells allows intoxication of predators that lack a cell surface receptor for the particular toxin, allowing bacteria-bearing exotoxins to kill a broader spectrum of predators, increasing the fitness of the otherwise 'defenceless' prey bacteria.

Invasion of endothelial cells and arthritogenic potential of endocarditis-associated Corynebacterium diphtheriae.

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.

[The ability of diphtheria causative agent to form biofilm].

The article deals with results of studying diphtheria causative agent capacities to form biofilm as one of mechanisms of persistence in human organism. The study object was strain of C.diphtheriae gravis tox+ obtained from nasopharynx of patient aged 19 in municipal hospital No 1 of town of Gukovo of Rostov oblast in 2011. The patient had diagnosis of "diphtheria of nasopharynx, typical filmy, localized, mild severity, even course". The control was implemented using the museum strain C.diphtheriae gravis tox+ No 665 from the L.A. Tarasevitch state research institute of standardization and biologic preparations control. It is established that diphtheria causative agent as an ability to form biofilm. The intensity of process of formation of exopolysaccharide is higher on glass that on plastic surfaces. The differences in degree of intensity of formation of biofilm are revealed between the strain circulation in population and museum strains C.diphtheriae gravis tox+. The vital capacity of biofilm forming microorganisms is related with adaptation possibilities of strains.

SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

Contour and persistence length of Corynebacterium diphtheriae pili by atomic force microscopy.

Many bacteria are characterized by nanoscale ultrastructures, for example S-layers, flagella, fimbriae, or pili. The last two are especially important for attachment to different abiotic and biotic surfaces and for host-pathogen interactions. In this study, we investigated the geometric and elastic properties of pili of different Corynebacterium diphtheriae strains by atomic force microscopy (AFM). We performed quantitative contour-length analysis of bacterial pili and found that the visible contour length of the pili can be described by a log-normal distribution. Our data revealed significant strain-specific variations in the mean visible contour length of the pili, ranging from 260 to 1,590 nm. To estimate their full contour length, which is not directly accessible from the AFM images, we developed a simple correction model. Using this model, we determined the mean full contour length as 510-2,060 nm. To obtain the persistence length we used two different methods of analysis, one based on the end-to-end distance of the pili and one based on the bending angles of short segments. In comparison, the bending angle analysis proved to be more precise and resulted in persistence lengths in the narrow range of 220-280 nm, with no significant strain-specific variations. This is small compared with some other bacterial polymers, for example type IV pili, F-pili, or flagella.

Strain-dependent arthritogenic potential of the zoonotic pathogen Corynebacterium ulcerans.

During the last decade the majority of diphtheria cases in Europe had Corynebacterium ulcerans as the etiologic agent with dogs and cats as the reservoir hosts. However, little has been documented about the virulence factors of this zoonotic pathogen. To set up an in vivo experimental C. ulcerans infection model, conventional Swiss Webster mice were intravenously infected with different doses (from 1 × 10(7) to 5 × 10(9) bacteria per mouse) of C. ulcerans strains, namely 809 (from human lower respiratory tract), BR-AD22 (from asymptomatic dog nares) and CDC-KC279. Mortality rates were demonstrated by LD(50) values ranging from 1.9 × 10(8) to 1.3 × 10(9). Viable bacteria were recovered from blood, kidneys, liver, spleen and joints. For CDC-KC279 and 809 strains (2 × 10(8)mL(-1)) approximately 85% and 72% of animals with articular lesions were observed, respectively; BR-AD22-infected mice showed no signs of arthritis. CDC-KC279 and 809 strains exhibited higher arthritogenic potential when compared to the homologous toxigenic (ATCC27012) and non-toxigenic (ATCC27010) strains of Corynebacterium diphtheriae. A high number of affected joints and arthritis index in addition to the histopathological features, including subcutaneous edema, inflammatory infiltrate, damage to bone tissue and synoviocyte hypertrophy, indicated a strain-dependent ability of C. ulcerans strains to cause severe polyarthritis. A correlation between the arthritis index and systemic levels of IL-6 and TNF-α was observed for C. ulcerans strains, with the exception of the non-arthritogenic BR-AD22 strain. In conclusion, C. ulcerans revealed a strain-dependent arthritogenic potential independent of DNAse, PLD and diphtheria toxin production.

Adhesion by pathogenic corynebacteria.

Pathogenic members of the genus Corynebacterium cause a wide range of serious infections in humans including diphtheria. Adhesion to host cells is a crucial step during infection. In Corynebacterium diphtheriae, adhesion is mediated primarily by filamentous structures called pili or fimbriae that are covalently attached to the bacterial cell wall. C. diphtheriae produces three distinct pilus structures, SpaA-, SpaD- and SpaH-type pili. Similar to other types, the prototype SpaA pilus consists of SpaA forming the pilus shaft and two minor pilins SpaB and SpaC located at the base and at the tip, respectively. The minor pilins SpaB/SpaC are critical for bacterial binding to human pharyngeal cells, and thus represent the major adhesins of corynebacteria. Like pili of many other gram-positive microbes, the assembly of corynebacterial pili occurs by a two-step mechanism, whereby pilins are covalently polymerized by a transpeptidase enzyme named pilin-specific sortase and the generated pilus polymer is subsequently anchored to the cell wall peptidoglycan via the base pilin by the housekeeping sortase or a non-polymerizing sortase. This chapter reviews the current knowledge of corynebacterial adhesion, with a specific focus on pilus structures, their assembly, and the mechanism of adhesion mediated by pili.

Detection of ascitic fluid infections in patients with liver cirrhosis and ascites.

BACKGROUND AND STUDY AIMS: Ascitic fluid infections (AFIs) are the frequent complications of advanced liver disease. Bacterial translocation is considered a key step in the pathogenesis of gut-derived bacterial infections; mainly spontaneous bacterial peritonitis (SBP) in cirrhotic patients. Bacterial DNA (bactDNA) in ascitic fluid and serum has been suggested as a surrogate marker for bacterial translocation. We attempted at the isolation and identification of bacteria in ascitic fluid in cirrhotic patients and the assessment of polymerase chain reaction (PCR) in ascitic fluid and serum. PATIENTS AND METHODS: Fifty cirrhotic patients having ascites with no signs of infection were included. Ascitic fluid cultures were obtained from patients. Ascitic fluid and serum were subjected to DNA extraction and PCR for the universal amplification of a region of the 16S ribosomal RNA (16S rRNA) gene to detect bactDNA. RESULTS: Bacteria were isolated from 9 (18%) of the ascitic fluid samples, and were mainly Gram-positive bacteria. BactDNA was detected simultaneously in the ascitic fluid and serum of 17 (34%) patients and in the ascitic fluid of only 2 patients. In a single patient with positive ascitic fluid culture no bactDNA was detected in ascitic fluid or serum. By considering AFIs as a positive ascitic fluid culture and/or the presence of bactDNA in the ascitic fluid and/or serum, ascitic fluid culture could detect 9 out of 20 patients with AFIs (45%), PCR of ascitic fluid could detect 19 out of 20 (95%) while PCR of serum could detect 17 out of 20 (85%). In 10 patients with culture negative non-neutrocytic ascites (CNNNA) bactDNA could be detected in serum and ascitic fluid. CONCLUSION: AFI can be caused by Gram positive as well as Gram negative organisms. A substantial percentage of cases with CNNNA show bactDNA in serum and ascitic fluid. PCR of ascitic fluid should, therefore, be used in the diagnostic workup of suspected cases of ascitic fluid infections.

Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae with host cells.

BACKGROUND: Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. RESULTS: Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. CONCLUSIONS: The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.