Salivary Mucins Play Active Role to Fight Cavities

WASHINGTON, DC - NOVEMBER 11, 2014 -- Salivary mucins, key components of mucus, actively protect the teeth from the cariogenic bacterium, Streptococcus mutans, according to research published ahead of print in Applied and Environmental Microbiology. The research suggests that bolstering native defenses might be a better way to fight dental caries than relying on exogenous materials, such as sealants and fluoride treatment, says first author Erica Shapiro Frenkel, of Harvard University, Cambridge, MA.

S. mutans attaches to teeth using sticky polymers that it produces, eventually forming a biofilm, a protected surface-associated bacterial community that is encased in secreted materials, says Frenkel. As S. mutans grows in the biofilm, it produces organic acids as metabolic byproducts that dissolve tooth enamel, which is the direct cause of cavities. “We focused on the effect of the salivary mucin, MUC5B on S. mutans attachment and biofilm formation because these are two key steps necessary for cavities to form,” says Frenkel.

“We found that salivary mucins don’t alter S. mutans’ growth or lead to bacterial killing over 24 hours,” says Frenkel. “Instead, they limit biofilm formation by keeping S.mutans suspended in the liquid medium. This is particularly significant for S. mutans because it only causes cavities when it is attached, or in a biofilm on the tooth’s surface.” She adds that the oral microbiome is better preserved when naturally occurring species aren’t killed. “The ideal situation is to simply attenuate bacterial virulence,” she says.

The study grew out of previous work in the investigators’ laboratory showing that other types of mucins, such as porcine gastric mucins, had protective effects against common lung pathogens, says Frenkel. With this in mind, they suspected that salivary mucins would play a protective role, but they were not sure what that would be.

“Defects in mucin production have been linked to common diseases such as asthma, cystic fibrosis, and ulcerative colitis,” says Frenkel. “There is increasing evidence that mucins aren’t just part of the mucus for structure or physical protection, but that they play an active role in protecting the host from pathogens and maintaining a healthy microbial environment. We wanted to apply these emerging ideas to a disease model that is a widespread, global public health problem—cavities. We chose to study the interaction of MUC5B with Streptococcus mutans because it is the primary cavity-causing bacteria in the oral cavity.”

The research makes a fundamental contribution to scientific understanding of host-microbe interactions, says principal investigator Katharina Ribbeck, of the Massachusetts Institute of Technology, Cambridge MA. “It is generating a paradigm shift from the textbook view of mucus as a simple catchall filter for particles, towards the understanding that mucus is a sophisticated bioactive material with powerful abilities to manipulate microbial behavior.”

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Mineralization of Sand Particles Boosts Microbial Water Filtration

WASHINGTON, DC – OCTOBER 10, 2014 - Mineral coatings on sand particles actually encourage microbial activity in the rapid sand filters that are used to treat groundwater for drinking, according to a paper published ahead of print in Applied and Environmental Microbiology. These findings resoundingly refute, for the first time, the conventional wisdom that the mineral deposits interfere with microbial colonization of the sand particles.

“We find an overwhelmingly positive effect of mineral deposits on microbial activity and density,” says corresponding author Barth F. Smets, of the Technical University of Denmark, Lyngby.

Mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. Coating certainly changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity and abundance of microbiota

Until now, rapid sand filters have been a bit of a black box, says first author Arda Gülay,one of Smets’ graduate students.

“In rapid sand filters, a combination of chemical, biological, and physical reactions help in the removal and precipitation of the impurities—iron, manganese, ammonia, and methane, for example,” says first author Arda Gülay,one of Smets’ graduate students.  In time, the sand filter grains become coated with minerals, much of which the system managers remove, periodically, via backwashing.

It turns out that the minerals form an abundant matrix around the sand particles, sort of honeycomb-like. “Bacterial cell density in these structures can be very high, and can be boosted further when extra ammonium is provided,” says Smets. The bacteria are normally engaged in removal of ammonium, manganese, and other impurities from the groundwater.

In fact, during the investigation, the ammonium-removal activity increased as the mineral deposits grew. “These positive mineral-microbe interactions suggest protective and supportive roles of the deposits,” says Smets. The investigators also measured a high diversity of ammonium and nitrite-oxidizing species.

The researchers’ direction involved a serendipitous twist. Early on, they discovered an unexpected positive correlation between the number of bacteria, and the degree of mineral coating of the sand particles, says Smets. “This was deemed worthy of further investigation, but we thought it would be a high risk effort. It was not until we saw actual cross sections of the mineral phases, which clearly reveal microbial cell like structures inside the deposits that we became aware of the unique discoveries we were making.”

A major question the research raises is whether the microbes influence the development of the microporosity, or simply take advantage of it, says Gülay. Either way, it could lead ultimately to steering the mineralization to create micro-structures designed to house microbial cells to perform specific functions.

The manuscript can be found online at http://bit.ly/asmtip1014a.  The final version of the article is scheduled for the November 2014 issue of Applied and Environmental Microbiology.

Zoos Exonerated in Baby Elephant Deaths; Data Support New Branch of Herpesvirus Family

WASHINGTON, DC – October 8, 2014 - Elephants are among the most intelligent non-humans, arguably on par with chimps, but both African and Asian elephants—separate species—are endangered. In 1995, 16-month old Kumari, the first Asian elephant born at the National Zoo in Washington, DC, died of a then-mysterious illness. In 1999, Gary Hayward of Johns Hopkins University and collaborators published their results identifying a novel herpesvirus, EEHV1 as the cause of Kumari’s sudden death. They now show that severe cases like this one are caused by viruses that normally infect the species, rather than by viruses that have jumped from African elephants, which was their original hypothesis. Hayward’s latest research appears ahead of print in two concurrently published papers in the Journal of Virology.

At the time of Kumari’s death, anti-zoo activists seized on the situation to call for abandoning all efforts to breed Asian elephants in zoos, as they claimed that zoos were spreading the deadly herpesvirus, says Hayward. Contrary to that, in the current research, “We showed that whereas some identical herpesvirus strains infected both healthy and diseased animals concurrently at particular facilities, the majority were different strains, and there has not been a single proven case of the same strain occurring at any two different facilities,” says Hayward. “Therefore, the viruses have not spread between zoos, and the sources of the viruses were most likely wild-born elephant herdmates. In fact, we also found the same disease in several Asian range countries, including in orphans and wild calves, and showed that the EEHV1 strains in India displayed the same genetic diversity as those in Western zoos.”

The papers also provide substantial data to support the hypothesis that the EEHV collectively represent a new, fourth major branch of the herpesvirus family, the proposed deltaherpesvirus subfamily (Deltaherpesvirinae), says Philip Pellett of Wayne State University, Detroit, who wrote an invited Commentary which accompanied Hayward’s papers. “Given that the three other branches were recognized over 30 years ago, establishment of a new subfamily would a big deal.”

Pellett adds that “Further scientific significance arises from the discovery of 12 new herpesviruses and identification of some new wrinkles in our understanding of herpesvirus diversity and evolution.”

In these studies, the investigators performed extensive DNA fingerprinting of the genetic signatures of all the known EEHV cases, as well as samples of EEHV virus that were obtained from wild Asian and African elephants, says Hayward. In the process, they identified seven different species of EEHVs and multiple different chimeric subtypes and strains of each.

“Because these viruses cannot be grown in cell culture, we had to develop sensitive and specific PCR techniques to be able to identify and compare the sequences of multiple segments of many different types of EEHV genomes directly from pathological blood and tissue DNA samples,” says Hayward. “Later, by also examining benign lung nodules from culled wild African elephants, we determined that EEHV2, EEHV3, EEHV6, and EEHV7 are natural endogenous viruses of African elephants, whereas EEHV1A, EEHV1B, EEHV4, and EEHV5 are apparently natural and nearly ubiquitous infections of Asian elephants that are occasionally shed in trunk washes and saliva of most healthy asymptomatic adult animals.”

Hayward notes that only one example of a lethal cross-species infection with EEHV3 into an Asian elephant calf has been observed, and that the viruses causing disease normally do so only in their natural hosts.

Close monitoring of Asian elephant calves in zoos has so far enabled life-saving treatment for at least nine infected Asian calves, says Hayward, suggesting that such monitoring may ultimately enable determining why some animals become susceptible to severe disease after their primary EEHV1 infections, while most do not. “About 20% of all Asian elephant calves are susceptible to hemorrhagic disease, whereas symptomatic disease is extremely rare in African elephant calves under the same zoo conditions,” says Hayward.

In another paper in the same issue of Journal of Virology, Hayward et al. demonstrate that the many highly diverged species and subtypes of EEHVs are ancient viruses that evolved separately from all other known subfamilies of mammalian herpesviruses within the ancestor of modern elephants, beginning about 100 million years ago.

Philip Pellett, of Wayne State University School of Medicine, Detroit, praises both of Hayward’s studies in this issue of the Journal of Virology: “The information gained in the new EEHV papers will be important for developing diagnostic tools for these viruses, and for developing therapeutic approaches to diseases caused by EEHV.”

Elephant populations have been plummeting. African elephants declined roughly from 10 million to half a million during the 20th century, due largely to habitat destruction, and intense poaching has since further decimated their numbers. Asian elephants, once in the millions, now number less than 50,000. They are threatened mostly by habitat fragmentation.

The full papers will appear in the December issue of the Journal of Virology.

Gut Bacteria Promotes Obesity in Mice

WASHINGTON, DC – September 30, 2014 – A species of gut bacteria called Clostridium ramosum, coupled with a high-fat diet, may cause animals to gain weight. The work is published this week in mBio®, the online open-access journal of the American Society for Microbiology.

A research team from the German Institute of Human Nutrition Potsdam-Rehbruecke in Nuthetal observed that mice harboring human gut bacteria including C. ramosum gained weight when fed a high-fat diet. Mice that did not have C. ramosum were less obese even when consuming a high-fat diet, and mice that had C. ramosum but consumed a low-fat diet also stayed lean.

Previous studies have found C. ramosum and other representatives of the Erysipelotrichi class in obese humans, said senior study author Michael Blaut, PhD, head of the institute’s Department of Gastrointestinal Microbiology. This suggests that growth of this organism in the digestive tract is stimulated by high-fat diets, which in turn improves nutrient uptake and enhances the effect of such diets on body weight and body fat.

“We were surprised that presence or absence of one species in a defined bacterial community affected body weight and body fat development in mice,” says Blaut.

Blaut and colleagues investigated the role of C. ramosum in three groups of mice: some harbored a simplified human intestinal microbiota (bacteria) of eight bacterial species including C. ramosum; some had simplified human intestinal microbiota except for C. ramosum, and some had C. ramosum only. The researchers called the first group SIHUMI, the second group SIHUMIw/oCra and the third group Cra. Mice were fed either a high-fat diet or low-fat diet for four weeks.

After four weeks eating a high-fat diet, the mouse groups did not differ in energy intake, diet digestibility, and selected markers of low-grade inflammation. However, SIHUMI mice and Cra mice fed a high-fat diet gained significantly more body weight and body fat, which implies that they converted food more efficiently to energy than did the SIHUMIw/oCra mice. By contrast, all groups of mice fed a low-fat diet stayed lean, indicating that the obesity effect of C. ramosum only occurred on high-fat diets.

The obese SIHUMI and Cra mice also had higher gene expression of glucose transporter 2 (Glut2), a protein that enables absorption of glucose and fructose, and fat transport proteins including fatty acid translocase (Cd36).

“Our results indicate that Clostridium ramosum improves nutrient uptake in the small intestine and thereby promotes obesity,” Blaut said. Associations between obesity and increased levels of lipopolysaccharides (components of the cell wall of gram-negative bacteria) causing inflammation, or increased formation of molecules called short chain fatty acids, reported by other researchers, were not found in this study, he said: “This possibly means that there is more than one mechanism underlying the promotion of obesity by intestinal bacteria.”

Through additional studies Blaut said he hopes to learn more about how C. ramosum affects its host’s energy metabolism and whether similar results occur in conventional mice given the bacteria. “Unraveling the underlying mechanism may help to develop new strategies in the prevention or treatment of obesity,” he said.

The current study was supported by the German Institute of Human Nutrition Potsdam-Rehbruecke.

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mBio® is an open access online journal published by the American Society for Microbiology to make microbiology research broadly accessible. The focus of the journal is on rapid publication of cutting-edge research spanning the entire spectrum of microbiology and related fields. It can be found online at http://mbio.asm.org.

The American Society for Microbiology is the largest single life science society, composed of over 39,000 scientists and health professionals. ASM's mission is to advance the microbiological sciences as a vehicle for understanding life processes and to apply and communicate this knowledge for the improvement of health and environmental and economic well-being worldwide.

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