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The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms.

ABSTRACT: In biofilms, bacteria that possess a negatively charged surface are embedded within a matrix of polymers consisting mainly of negatively charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively charged substance, for example, a basic protein, exists within biofilms to neutralize charge-charge repulsions and act as a 'glue' attaching negatively charged bacteria to negatively charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly abundant nucleoid-associated histone-like protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, for example, (a) availability in association with e-DNA; (b) multivalent DNA binding; (c) non-sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU-cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and noncanonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical-chemical, microscopy-based, and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS, that this facilitates DNA-DNA, DNA-LPS, and LPS-LPS interactions, and that this facilitates bacterial clumping and attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (nonshaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide, and move away from each other, in opposition to the accreting influence of HUs).

SUBMITTER: Thakur B

PROVIDER: S-EPMC8063757 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA-binding properties and other protein features.Performed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA-binding properties of HU from N. gonorrhoeae, which sequence is similar to one of E.coli HU, and HU from M. gallisepticum and S. melliferum which sequences are distant from E.coli protein. We found that in respect to dsDNA binding, only S. melliferum HU essentially differs from E.coli HU. In respect to binding of distorted DNA structures, S. melliferum HU and E.coli HU have similar properties but essentially different from M. gallisepticum HU and N. gonorrhea HU. We found that in respect to dsDNA binding, only S. melliferum HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than E.coli and N. gonorrhoeae. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.HU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding's even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from N. gonorrhoeae, M. gallisepticum and S. melliferum are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.

Project description:The oral cavity is home to a wide variety of bacterial species, both commensal, such as various streptococcal species, and pathogenic, such as Porphyromonas gingivalis, one of the main etiological agents of periodontal disease. Our understanding of how these bacteria ultimately cause disease is highly dependent upon understanding how they coexist and interact with one another in biofilm communities and the mechanisms by which biofilms are formed. Our research has demonstrated that the DNABII family of DNA-binding proteins are important components of the extracellular DNA (eDNA)-dependent matrix of bacterial biofilms and that sequestering these proteins via protein-specific antibodies results in the collapse of the biofilm structure and release of the resident bacteria. While the high degree of similarity among the DNABII family of proteins has allowed antibodies derived against specific DNABII proteins to disrupt biofilms formed by a wide range of bacterial pathogens, the DNABII proteins of P. gingivalis have proven to be antigenically distinct, allowing us to determine if we can use anti-P. gingivalis HU? antibodies to specifically target this species for removal from a mixed-species biofilm. Importantly, despite forming homotypic biofilms in vitro, P. gingivalis must enter preexisting biofilms in vivo in order to persist within the oral cavity. The data presented here indicate that antibodies derived against the P. gingivalis DNABII protein, HU?, reduce by half the amount of P. gingivalis organisms entering into preexisting biofilm formed by four oral streptococcal species. These results support our efforts to develop methods for preventing and treating periodontal disease.IMPORTANCE Periodontitis is one of the most prevalent chronic infections, affecting 40 to 50% of the population of the United States. The root cause of periodontitis is the presence of bacterial biofilms within the gingival space, with Porphyromonas gingivalis being strongly associated with the development of the disease. Periodontitis also increases the risk of secondary conditions and infections such as atherosclerosis and infective endocarditis caused by oral streptococci. To induce periodontitis, P. gingivalis needs to incorporate into preformed biofilms, with oral streptococci being important binding partners. Our research demonstrates that targeting DNABII proteins with an antibody disperses oral streptococcus biofilm and prevents P. gingivalis entry into oral streptococcus biofilm. These results suggest potential therapeutic treatments for endocarditis caused by streptococci as well as periodontitis.

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The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms.

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