Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics.

Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli.

We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His(6)) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His(6) and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

Targeted replacement of KV1.5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of I(K,slow) and resistance to drug-induced qt prolongation.

The K(+) channel mKv1.5 is thought to encode a 4-aminopyridine (4-AP)-sensitive component of the current I(K,slow) in the mouse heart. We used gene targeting to replace mKv1.5 with the 4-AP-insensitive channel rKv1.1 (SWAP mice) and directly test the role of Kv1.5 in the mouse ventricle. Kv1.5 RNA and protein were undetectable, rKv1.1 was expressed, and Kv2.1 protein was upregulated in homozygous SWAP hearts. The density of the K(+) current I(K,slow) (depolarizations to +40 mV, pA/pF) was similar in left ventricular myocytes isolated from SWAP homozygotes (17+/-1, n=27) and littermate controls (16+/-2, n=19). The densities and properties of I(peak), I(to,f), I(to,s), and I(ss) were also unchanged. In homozygous SWAP myocytes, the 50-micromol/L 4-AP-sensitive component of IK,slowwas absent (n=6), the density of the 20-mmol/L tetraethylammonium-sensitive component of I(K,slow) was increased (9+/-1 versus 5+/-1, P<0.05), and no 100- to 200-nmol/L alpha-dendrotoxin-sensitive current was found (n=8). APD(90) in SWAP myocytes was similar to controls at baseline but did not prolong in response to 30 micromol/L 4-AP. Similarly, QTc (ms) was not prolonged in anesthetized SWAP mice (64+/-2, homozygotes, n=9; 62+/-2, controls, n=9), and injection with 4-AP prolonged QTc only in controls (63+/-1, homozygotes; 72+/-2, controls; P<0.05). SWAP mice had no increase in arrhythmias during ambulatory telemetry monitoring. Thus, Kv1.5 encodes the 4-AP-sensitive component of I(K,slow) in the mouse ventricle and confers sensitivity to 4-AP-induced prolongation of APD and QTC: Compensatory upregulation of Kv2.1 may explain the phenotypic differences between SWAP mice and the previously described transgenic mice expressing a truncated dominant-negative Kv1.1 construct.