Type IV Pili and the CcpA Protein Are Needed for Maximal Biofilm Formation by the Gram-Positive Anaerobic Pathogen Clostridium perfringens▿

The predominant organizational state of bacteria in nature is biofilms. Biofilms have been shown to increase bacterial resistance to a variety of stresses. We demonstrate for the first time that the anaerobic gram-positive pathogen Clostridium perfringens forms biofilms. At the same concentration of glucose in the medium, optimal biofilm formation depended on a functional CcpA protein. While the ratio of biofilm to planktonic growth was higher in the wild type than in a ccpA mutant strain in middle to late stages of biofilm development, the bacteria shifted from a predominantly biofilm state to planktonic growth as the concentration of glucose in the medium increased in a CcpA-independent manner. As is the case in some gram-negative bacteria, type IV pilus (TFP)-dependent gliding motility was necessary for efficient biofilm formation, as demonstrated by laser confocal and electron microscopy. However, TFP were not associated with the bacteria in the biofilm but with the extracellular matrix. Biofilms afforded C. perfringens protection from environmental stress, including exposure to atmospheric oxygen for 6 h and 24 h and to 10 mM H2O2 for 5 min. Biofilm cells also showed 5- to 15-fold-increased survival over planktonic cells after exposure to 20 μg/ml (27 times the MIC) of penicillin G for 6 h and 24 h, respectively. These results indicate C. perfringens biofilms play an important role in the persistence of the bacteria in response to environmental stress and that they may be a factor in diseases, such as antibiotic-associated diarrhea and gas gangrene, that are caused by C. perfringens

NanR, a transcriptional regulator that binds to the promoters of genes involved in sialic acid metabolism in the anaerobic pathogen clostridium perfringens

Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell

Gel mobility shift experiments with NanR and the <i>nanI</i> and <i>nanE</i> promoter regions.

<p>Gel mobility shift assays with the <i>nanE</i><b>(A)</b> and <i>nanI</i><b>(B)</b> promoter regions that were PCR amplified as described in the Materials and Methods. The binding reactions shown in panel B were incubated under the same conditions as those in A. In (A) and (B) the panels on the right show the results of binding competition experiments using unlabeled target DNA as indicated.</p

The <i>nanI</i> promoter from strain 13(pSM230) shows sialic acid-inducible activity.

<p>β-glucuronidase activity was measured for each strain indicated. Strain AH1 is a <b>β</b>-glucuronidase mutant derived from strain 13 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133217#pone.0133217.ref031" target="_blank">31</a>]. Values shown are the mean and SEM of triplicate biological samples. The <i>P</i> value, calculated using the student's two-tailed t-test, is shown in the figure.</p

Strains, plasmids and primers.

<p>Strains, plasmids and primers.</p

Identification of transcription starts sites upstream of <i>nanI</i> and <i>nanJ</i>.

<p><b>(A)</b>. Primer extension results on RNA isolated from <i>C</i>. <i>perfringens</i> strain 13 using a primer specific for <i>nanI</i> mRNA. The cells were grown on PY (-) or PY plus 1 mg/ml Neu5Ac (+). The GATC lanes show the results of sequencing using the same primer used for the primer extension experiments. The arrows labeled P1, P2, and P3 indicate the position of the putative start sites. The location of P1, P2, P3 and conserved 14 bp elements are shown in the <i>nanI</i> promoter sequence. <b>(B)</b>. Primer extension results for <i>nanJ</i> from <i>C</i>. <i>perfringens</i> strain 13. The graph shows peak fluorescence of the 5’- 6 FAM labeled RT-PCR product for <i>nanJ</i>. Numbers below each peak indicate the length of the product and the peaks on the bottom line show the location of size standards used to estimate the length of the transcripts. The cells were grown on PY (-SA) or PY plus 1 mg/ml Neu5Ac (+SA). The location of P1, P2 and a conserved 14 bp element are shown in the <i>nanJ</i> promoter sequence.</p

Alignment of conserved 14-bp elements located in the promoter regions of sialic acid-related genes.

<p>Majority refers to the consensus sequence calculated by the Megalign program (Lasergene, v. 11, DNASTAR). “Position” refers to the distance from the adenosine in the initiator methionine residue for each gene shown to the left. The numbers following each gene indicate the proximity of the sequence to the start codon with the sequences closest to the start represented by the lowest number.</p

Extracellular sialidase activity of wild-type <i>C</i>. <i>perfringens</i>, BT1 (<i>nanI</i>), BT2 (<i>nanJ</i>), and BT3 (<i>nanInanJ</i>).

<p>Cells were grown overnight in 5 ml of PY with (+) or without (-) 1 mg/ml sialic acid as indicated. Sialidase specific activity was defined as μmoles of 4-methylumbelliferyl-<b>α</b>-D-N-acetylneuraminic acid hydrolyzed per minute per milligram of protein. Values shown are the mean and SEM of triplicate biological samples. The <i>P</i> values, calculated using the student's two-tailed t-test, are shown in the figure.</p