Source: https://mbioblog.asm.org/mbiosphere/2015/08/index.html
Timestamp: 2019-04-19 08:46:37+00:00

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The bacterium that causes cholera, Vibrio cholerae, can grow both aquatically and in a human host. To survive, the bacterium must be able to live in very distinct conditions: imagine how different the temperature, available nutrients, and neighboring microbes are between these two niches. To adapt, V. cholerae must turn on and off the genes appropriate for its current environment. How does the bacterial cell know what its current environment is and which genes it needs?
This is the question being addressed by Dr. Jim Bina and graduate student Vanessa Ante. Their research focuses on the questions: what are the environmental cues, and how do these cues affect gene expression in V. cholerae? Ante’s recently published findings in the Journal of Bacteriology demonstrate that a component of the human intestine, bile salt, acts as one of the environmental cues to activate expression of virulence-related gene, leuO.
Bile is secreted by the liver into the small intestine, and contains a number of antimicrobial factors. The major component is bile acids, such as deoxycholate, cholate, and chenodeoxycholate. These detergent-like molecules are important for digestion and can also disrupt the microbial membrane – but Ante’s work shows they can be an alert to V. cholerae. As V. cholerae is ingested from contaminated water, it passes into the gastrointestinal tract. “Because we have bile salts present in the small intestines, these bile salts act as a signaling molecule, with ToxR responding and regulating LeuO in response,” explains Ante. ToxR and LeuO are two cholera proteins that play an important role in detecting environmental cues.
Bina adds: “It's been known for a long time that ToxR, a membrane-associated regulator, has a domain in the periplasm of the organism that is thought to be a sensing domain.” After sensing environmental molecules, the ToxR sends a signal to the DNA binding domain, which can then regulate the target genes. ToxR signaling has several arms: regulating expression of virulence factors, such as cholera toxin, and regulating expression of porin proteins that are important for bile resistance (the latter is not pictured in the image at right).
“What Vanessa has shown is that in addition to porin protein, in the presence of bile, ToxR also turns on LeuO. LeuO goes on and regulates a number of downstream effects and also contributes to bile resistance, suggesting that the regulatory cascade being controlled by ToxR to adapt to the environment of the intestine is broader than just these porin proteins,” explains Bina. “What this paper is showing is that one of the environmental signals turns out to be bile acids – at least in the test tube,” he continues, and that ToxR “is necessary for sensing bile acids and then turning on the downstream effects,” including bile resistance.
The research adds to the known targets of ToxR signaling. Further, it shows that LeuO activation doesn’t require an intermediary protein. “ToxR can regulate a number of genes, and the major one is ToxT, which then goes on and regulates virulence factor production. We show that ToxR is able to directly bind to LeuO without ToxT being involved,” says Ante. This research shows “the role of ToxR, at least in adaptation to the environment in the intestine, is broader than what was previously appreciated,” says Bina.
The Bina lab had previously shown that ToxR regulates LeuO when cyclic dipeptides are present. When cyclic dipeptides, which are produced by a number of bacteria, are the activating molecule, LeuO activation occurs with simultaneous decrease of virulence factor genes. However, bile salt activation of LeuO did not have a similar decrease of virulence factor genes. Why not?
Input: environmental cue. Output: proper gene expression. As the above toxin regulation schematic suggests, the circuit is rarely linear. Understanding these complex circuits allows researchers to understand how bacteria sense and adapt to their current environment. In the case of pathogens like V. cholerae, these circuits can also identify potential drug targets, if a given drug throws a wrench in the circuitry gears. Researchers have previously described small molecule inhibitors of ToxT transcription – just the sort of wrench to stop V. cholerae from adjusting to its environment and producing toxin. But before the circuit can be interrupted, it must first be fully understood. The story of bile salt stimulation of ToxR-LeuO moves us one step closer to that goal.
One of the most successful battles in the war on HIV has been stemming mother-to-child transmission of the virus. By treating expectant mothers with antiretrovirals that limit the virus numbers in the blood, HIV-infected women are able to have healthy, uninfected babies. However, babies are still susceptible to HIV virions that can be found in breastmilk, and breastfeeding is not recommended for new mothers in the U.S. Since these recommendations were made in the 1990s, infections passed to newborns right after birth have decreased 90% in the U.S.
However, HIV screening and antiretrovirals aren’t available worldwide, and the numbers reflect this fact: nearly 10% of new HIV-1 infections are in infants. Some infants are unfortunately exposed to HIV during pregnancy, birth, or breastfeeding. Around 40% of exposed babies contract HIV infection – but that means nearly half do not. In fact, of uninfected, breastfeeding babies with infected mothers, only 15% contract HIV infection through their mother’s milk. Two new research papers in the Journal of Virology address the question: why do some breastfeeding babies contract HIV but others don’t?
One way to approach the question is to examine breast milk from infected mothers. Is there something in breast milk that might protect newborns from infection? This was the question posed by a team of physicians and researchers led by Dr. Sallie Permar. They used statistical modeling to explore associations between protection and milk components (such as antibody levels). They found no association between postnatal transmission risk and immune characteristics of the breast milk, such as HIV-1 envelop-specific IgG antibodies, antibody-dependent cell-mediated cytotoxicity, or neutralization activity.
What the team did find is an association between IgA antibodies and reduced transmission risk – specifically, IgA antibodies against HIV-1 envelope gp140. IgA play an important role in mucosal immunity, and therefore it may strongly influence neonate viral susceptibility. Characterizing these IgA antibodies, together with work previously published from the group on other antibodies and antiviral components found in breast milk, may help reduce mother-to-child transmission globally and lead to candidates for an infant vaccine.
A second team of researchers, led by Dr. J Victor Garcia-Martinez, have also published findings on the anti-HIV properties of breast milk. They wanted to look at the anti-HIV properties of HIV-infected mothers (the milk from uninfected mothers is known to be antiviral). This team used a mouse model of oral HIV transmission to ask what type of breast milk contains anti-viral qualities. They found that milk from both HIV-positive and -negative women was able to decrease HIV transmission, regardless of whether the infected women had transmitted HIV to their newborns.
From these results, the researchers conclusively support the current recommendations that HIV-positive mothers in resource-limited areas breastfeed in combination with HIV treatment. In resource-poor regions, breast milk is the best option for feeding newborns, since it is both nutrient rich and hydrating. While the chance of passing the disease to one’s child remains, the antiviral properties of breast milk decrease this risk, especially in concert with antiviral drugs.
These results suggest breast milk, even from HIV-infected mothers, contains immunoprotective components that protect neonates from HIV transmission. Further characterization of these components may lead to even better protection of infant and possibly adult populations, if the components protect from other routes of transmission.
Roughly 75% of all new, emerging, or re-emerging diseases affecting humans at the beginning of the 21st century are zoonotic diseases, meaning they originated in animals. For the past several years, researchers with the United States Agency for International Development’s PREDICT project have been working to discover and characterize viruses at the wildlife-human interface in order to be better prepared against future epidemics.
"We want to know what’s out there, and we want to try and understand the factors or the ‘drivers’ that cause disease emergence,” said Simon Anthony, D.Phil, assistant professor, Center for Infection and Immunity, Columbia University, New York City, who is involved with PREDICT. “Most of the viruses that exist in wildlife will never spillover into people or cause disease, but we want to be prepared and learn how to identify those that pose the greatest risk."
Examples of headline grabbing zoonotic diseases include HIV, which originated from primates in Africa, and severe acute respiratory syndrome (SARS), which originated from civets and other animals. HIV is an ongoing public health problem and the SARS global outbreak of 2003 spread to more than two dozen countries in North America, South America, Europe, and Asia before being contained.
Learning how to prevent future epidemics can sometimes mean having to look at events that occurred in the past. “We try to understand the pathways that previously led to successful spillover and disease emergence in order to identify those that may lead to new emergence in the future,” said Dr. Anthony.
In a recent discovery, researchers identified a virus in seals that is related to human hepatitis A. The discovery was made while scientists were investigating the dying off of seals in New England. In 2011, dead harbor seals started appearing on the coast of New England. An investigation determined that a deadly strain of avian influenza was the culprit. In an effort to determine what other viruses might be co-occurring with the influenza, researchers performed deep sequencing of all the viruses present in three of the dead harbor seals and discovered the new virus, which they are calling phopivirus. An analysis of additional seals living off the coast of New England (29 harbor seals, 6 harp seals and 2 grey seals) identified phopivirus in seven more marine mammals. Prior to this work, the hepatitis A virus was the only known member of a group called the ‘hepatoviruses’, so-called because of their preference for infecting the liver of their hosts. Humans and non-human primates were the only known hosts.
Various factors, including the fact that the virus was found in different species of seals, suggest that the virus has been present in seals for a fairly long time. The researchers next plan to look at species that have close interactions with seals to see if they can find other wildlife reservoirs of hepatitis A-like viruses. "Coyotes regularly prey upon live seals and scavenge carcasses along the coast, so it would be very interesting to examine coyotes to see if they have any similar viruses," said Katie Pugliares, MS, a senior biologist at the New England Aquarium in Boston, who was also involved in the study.
We’re approaching the end of summer, and many mBiosphere readers may want to take advantage of the hot weather to go hiking and camping before the chill of autumn sets in. But be prepared – in certain parts of North America, hikers are advised to wear protective clothing to help avoid tick bites, which may carry the causative agent of Lyme Disease, Borrelia burgdorferi. Where did this bacterium come from and why does it infect humans? In Applied and Environmental Microbiology this week, a new review by Dr. Nicholas Ogden et. al. covers the evolutionary and geographical history of Lyme Disease in North America.
B. burgdorferi was discovered in the early 1980s as the causative agent of Lyme Disease (in other parts of the world, B. afzelii and B. garinii can spread a similar disease). This bacterium is carried by hard-bodied ticks, which feed on deer and rodents living in temperate woody environments. The bacterium doesn’t require humans as part of its life cycle (unlike other zoonotic infections), and people are considered a “dead-end” host for this disease. The number of Lyme Disease diagnoses has increased each year since its emergence in the 1970s to over 300,000 annual cases in the US; this number will likely increase, as habitat and climate changes have increased the tick populations in the more heavily populated regions of southern Canada. Genetics suggests the tick migration into Canadian territory will mimic patterns in US locations directly south, since many of these ticks are thought to move on migratory birds traveling between the two countries.
There are 111 sequence types (ST) of B. burgdorferi, with distinct geographical locations (see Figure, taken from this article on B. burgdorferi sequence types). Some of these strains may be better at infecting people (and other hosts) than others, but B. burgdorferi appears to have a wider range of host animals than other Borrelia species. Does infection with these different STs manifest in different disease symptoms?
In general, there are three stages of Lyme Disease. In Early Localized stage, inflammation around the initial tick bite produces the characteristic “bullseye” pattern. As the bacteria spread from this initial infection site, Early Disseminated infection may lead to symptoms ranging from rash to neurological problems. If left untreated, patients may have severe nervous or cardiac system consequences. Unlike most other bacterial disease, no known virulence factors are associated with disease; the best hypothesis at the moment is that the disease sympotoms are due to inflammation and subsequent auto-antibodies. Because of this, pathogenicity is associated with the ability of the bacterium to disseminate and persist, although some genetic determinants have yet been identified that correlate with severity of disease.
One of these genetic determinants is ospC gene, which has been categorized into major allele groups. OspC is a surface lipoprotein required for mammalian infection. Epidemiological studies have shown some groups (A, B, H, I, and K) are more often associated with hematogenous dissemination early in the course of Lyme Disease, while others, such as T and U, are more likely to cause EM lesions alone and non-disseminated infection.
The association of ospC allelic groups with greater pathogenicity in people suggests a selection of this characteristic, despite the fact that people are dispensable for the B. burgdorferi lifecycle. This pathogenic phenotype isn’t necessarily reflective of a virulence selection. For example: in some rodent hosts, B. burgdorferi may maintain bacterial infection, which allows future transfer of bacterial infection to new ticks, continuing the cycle of infection. However, other rodents are infected only transiently before elimination by the host immune response. This is related to the bacterial ST, with the ST of long-lasting rodent infections more likely to cause disseminated disease in people. Thus, selection for virulence in people seems to be a byproduct of a selection for increased transmission from a nonhuman host.
If researchers are able to find a pattern in Lyme Disease outbreaks and B. burgdorferi strains, it will help public health advocates better warn citizens of potential dangers. Imagine a trail closed due to high Lyme Disease risk, similar to beaches closed due to high E. coli numbers. Epidemiologists might be able to predict where future outbreaks will occur, based on migratory patterns of birds and deer. While we don’t have these answers just yet, we’ve learned a lot in the past three decades. With the rapid advancement of sequencing and other technologies, scientists’ abilities to address these issues hope to come sooner, rather than later!
Streptococcus species are a natural part of the microflora: its ability to survive under a variety of conditions allow it to colonize several niches on the human body. Some of these species live as mostly harmless commensals, while some of these are potential pathogens that can cause disease under the right circumstances. Group B Streptococcus (GBS), or Streptococcus agalactiae, falls under both categories, as healthy adults rarely present GBS disease symptoms, but those with immunocompromised immune systems – especially the very young and the elderly – can be susceptible to invasive disease.
GBS are resident bacteria of the gastrointestinal tract and the genitourinary tract in 25% of women. The danger comes when GBS are passed to an infant during birth. The still-developing immune system of newborns makes them susceptible to GBS infection, and GBS is the leading cause of meningitis and sepsis in young infants (under 3 months old). Because of this, women testing positive for GBS are given antibiotics during labor, to decrease (but not eliminate) the chance of passing on this potentially deadly microbe. However, GBS persists as a threat to young infants, with 29,000 cases and 1750 deaths in the U.S. in 2013.
Scientists have wondered whether transferred GBS from a mother to her child needs to adapt to the environmental changes associated with a new host. Specifically, are there genetic differences between the carriage and disease states? To test this, Alexandre Almeida and Dr. Philippe Glaser sequenced the genome of GBS isolated from 19 infected newborn infants and their mothers.
GBS is an encapsulated bacterium, and its capsule is one of the major virulence factors facilitating disease. Bacterial strains are categorized by their capsule type and genotype. In their research, Almeida et. al. found that most of the strains isolated (66%) had a capsule variation strongly associated with virulence (serotype III), and half of these serotype III isolates were in a well-characterized hypervirulent genotype (ST-17). All mother-child isolates shared the same capsule type, and only two pairs differed in genotype. In these two cases, the child was colonized with the ST-17 genotype while the mothers contained two different, less virulent genotypes.
By investigating mutations among the entire genomic sequence, the scientific team found eight mother-child pairs with no differences in genomic sequence. These identical strains were nevertheless more pathogenic in the neonates than in their mothers. The nine mother-child pairs that did differ had differences that manifested both as changes in single nucleotides (SNPs) and insertions or deletions of nucleotides (INDELs). In one case, multiple strains were detected in the digestive system of a child, but only one in that same child’s blood. The blood-born strain contained four mutations, one in the upstream region of the gene for CovR, a response regulator that activates other virulence-related genes. When comparing gene expression between strains from this mother-child pair, no difference in covR gene expression levels was detected, but CovR-regulated gene expression was higher in the child’s isolates. One possible scenario is that this bloodstream strain was selected for because of its increased virulence, but another possibility is that a mutation in one of the non-invasive strains somehow allowed the bloodstream strain to become systemic.
Most of the identified mutations had not been found in any of the 300 GBS genomes currently available. Several of these mutations were in noncoding regions, such as the upstream region of the covR gene. This type of mutation may play an important role in changing the amount of gene expression, and thus the virulence associated with the strain.
Finally, by taking samples at a later time point, the researchers found that three mothers were colonized with infant strains shortly after birth. This may point to breastfeeding as a means for infants to transfer their virulence-adapted strains back to their mothers (this phenomenon is best known with Candida albicans oral infection, or thrush). Do these virulence-adapted strains undergo selection back toward commensalism? Experiments to look at the change in populations over time may determine the selective pressure for Group B Strep adaptation in different host environments.
Should infectious disease researchers be allowed to generate lab strains that are more pathogenic than naturally occurring microbes?
That question, and the issues that surround it, is at the heart of the gain-of-function (GOF) debates ongoing among microbiologists, particularly in the virology community. On the one hand, selecting for pathogenic traits that increase microbial fitness may allow doctors to generate a more effective vaccine or epidemiologists to lookout for particular genetic markers. On the other, scientists may misplace or accidentally release dangerous strains.
The last point is not a trivial one, since despite the strong regulations around pathogenic microbes, accidental lab infections do occur. But have any of these infections been spread beyond the immediate lab personnel? This is one aspect being investigated in a new report from mBio by Dr. Michelle Rozo and Dr. Gigi Kwik Gronvall, in regards to the 1977-78 influenza season. “The fact that the 1977 influenza season was not caused by nature – it was clearly man-made – is not generally appreciated, even among scientific circles,” says Gronvall.
Its similarity to a strain that had circulated a quarter-century prior raised suspicions about the origins of this influenza virus. Could this strain have been deliberately spread as a bioterrorism attack? Is it possibly a flu vaccine strain that wasn’t properly attenuated? Or could this strain have escaped from a biomedical research facility, possibly through an accidental lab-acquired infection?
This last point is one that has been picked up by those arguing against the right of scientists to perform GOF studies. If a strain with normal-to-lesser pathogenicity is still able to encircle the globe, imagine the ramifications if the strain had been highly pathogenic! In fact, it was influenza research that sparked the recent GOF debates a few years ago.
Gronvall and her colleague Michelle Rozo looked at the 1977 flu sequence and compared it to sequences of virus sequences from the late 1950s. These confirmed the relatedness of these strains and added support to this strain being a man-made event. However, there was no support in this data that the event occurred due to a lab accident, and in fact, evidence supports another possible explanation: a flawed vaccine trial.
“While we can't say for sure without a lot more historical data, the bulk of the evidence favors the vaccine trial explanation,” Gronvall explains. “Many of the flu samples were temperature sensitive, suggesting laboratory manipulation in preparation for a vaccine. There was interest in the H1N1 flu after a small outbreak of a virus of the same type in 1976 (the swine flu case), so vaccine preparation was likely initiated as a result.
“At that time, there was a lot of development of live-attenuated flu vaccines, but techniques were pretty crude compared to what is done today, and in many cases these weakened vaccine strains could revert and cause disease. Also, a renowned virologist from China -- where the outbreak likely started-- also communicated that he thought the virus was a result of vaccine trials done using several thousand military recruits.
What does this mean for the future of GOF research?
“Given that it was likely the result of a vaccine trial that went awry, the 1977 flu epidemic should not be used as a cautionary tale for GOF research,” concludes the author.
Lignocellulosic plant biomass is one of the most available and renewable materials on earth. It serves as a potent source of energy, being comprised of several polysaccharide sugars crosslinked to lignin. Sugars fermented into bioethanol can be used as a fuel source or additive. But removing the polysaccharides and breaking them down into smaller sugar subunits requires heat, chemical, and enzymatic treatment, and is energetically and financially expensive. Research on a wide variety of lignocellulose-degrading microbes, from archaea to bacteria to fungi, is therefore focused on finding a more efficient way to degrade these materials.
Enter Caldicellulosiruptor bescii. This organism is able to decompose lignocellulosic biomass anaerobically at high temperature, an ability researchers hope to harness to bypass costly thermochemical treatment. As a thermophile, the bacteria can survive at high temperature, a condition that already facilitates polysaccharide breakdown. Understanding this bacterial metabolic process could lead to more efficient biofuel production.
While analyzing the C. bescii genome, Israel Scott, working in Dr. Michael Adams’ lab, noticed an unusual finding: genes encoding a tungstate transporter. This was notable because utilization of the heavy metal tungsten is extremely rare in biological systems.
The researchers wanted to know if the genes were indeed importing tungsten, or if a similar, more commonly used element like molybdenum might be imported. The bacterium Pyrococcus furiosus has several characterized tungsten-utilizing, aldehyde-oxidoreductase enzymes (termed AOR). The P. furiosus enzyme was expressed in C. bescii, where it would only function if tungsten was being imported. After finding that the P. furiosus enzymes indeed functioned, the C. bescii homologs were identified and characterized.
AOR enzymes from P. furiosus have a range of aldehyde substrates (formaldehyde, propionaldehyde, crotonaldehyde, glutaraldehyde, isovaleraldehyde, benzaldehyde, and glyceraldehyde-3-phosphate), but the C. bescii strain was unable to oxidize any of these, leading the researchers to name the C. bescii homolog XOR, for “x”-oxidizing, and the divergence of XOR from the AOR family was confirmed through phylogenetic analyses.
The bacterium highly expresses XOR (it constitutes 2% of the cytoplasmic protein contents), which appears to be the only tungsten-containing enzyme in the genome. Attempts to make a gene deletion mutant were unsuccessful, suggesting that whatever substrate XOR degrades, it is an essential part of the bacterial metabolism.
Defining the enzymatic substrate will help scientists better utilize the unusual metabolism of C. bescii. AOR from P. furiosus can be utilized to generate alcohol from organic acids; maybe C. bescii can be similarly used. If we can better understand how C. bescii uses tungsten-containing enzyme to break down ligninocellulose, we can move closer to using its activity for bioengineered fuels.
HA is a glycoprotein found in the viral envelope of influenza A virus as a trimer (see schematic at left). HA is required for the first step of the viral life cycle: attachment of the virus to its new host cell. After HA binds its sialic acid receptor, the host takes up the virus via endocytosis. As the endosome becomes acidic, HA changes shape to insert into the lipid membrane, fusing viral and endosomal membranes to release viral genomic contents into the cytoplasm (see schematic below). Because HA is so important for these steps in viral replications, understanding how it functions is important to understanding influenza biology.
Drs. Wei Wang and Carol Weiss wanted to look at how HA sequence affects its stability. There are several major domains in the HA protein: the surface used for attachment (HA1, yellow in the diagram at right), the coiled-coil domain able to change conformations in the endosome (HA2, blue), the fusion peptide and the fusion peptide pocket. They looked at the HA from the 2009 pandemic H1N1 strain, as well as two seasonal H1N1 strains.
By combining the HA1-HA2 portions from each virus, the researchers found HA1 from the pandemic virus combined with seasonal HA2 didn’t fuse efficiently. When looking at the HA sequences, they found a difference in an interacting amino acid pair between the HA1/HA2 of the different strains. In the pandemic HA, an arginine and histidine (R-H) interaction was paired between HA1/HA2 domains, while the seasonal HA contained a histidine and lysine (H-K) interaction at these sequences (these interactions are marked with arrows in the above left diagram). The chimera contained one of each: the pandemic R and the seasonal K. Did the R-K pairing destroy HA function?
Mutational analyses was used to address this question. The results revealed the importance of histidine at this interaction site for HA function (on either the HA1 or HA2 domain). If either HA sequence was engineered to change the histidine residue, both the acid sensitivity and protein function changed. Without histidine, the fusion occurred at higher or lower pH than with histidine. Although there was a difference in fusion, there was no difference in any of the HA molecules in attachment.
These studies identified key residues that play a role in HA stability, and therefore are important for HA function. They also demonstrate a constraint in HA mutation: when the researchers investigated HA genetically altered to have a various non-histidine amino acid pairings, they found a decrease in infectivity, regardless of whether the changes increased or decreased acid stability and fusion capabilities. Since pH decreases as endosomes mature, this suggests that HA fusion timing is an important aspect of influenza viral fitness, and that mutations at this site (in H1 influenza virus) due to antigenic drift would be less infectious.
By comparing available sequences, the researchers further found most (>90%) of human seasonal influenza H1 HA isolates contains a histidine residue as an H-K pairing at these residues, while most (>95%) avian isolates have a lysine-aspargine (K-N) pairing. The research from Wang et. al., now published in the Journal of Virology, not only contributes to a greater understanding of how HA functions, but provides a molecular tool to identify potential crossover sequences. Identifying avian H1 HA sequences with histidine at this interaction site may help find viruses better prepared to survive a jump into humans.
Targeted drug delivery is the ultimate goal: imagine fewer side effects, more selective killing, and lower drug concentration needed to reach a desired effect. Toward that end, microbes are well ahead of us (as usual), having developed several targeted delivery systems.
One example of these targeted delivery systems are the A-B toxins. A-B toxins have at least two different protein parts: The B protein part confers specificity by binding to a cellular receptor and triggering endocytosis of the toxin. The A protein part confers activity – usually toxicity – by interfering with a cellular enzyme or having enzymatic activity of its own. In the case of one famous A-B toxin, anthrax toxin, the B component binds to the cellular receptor ATR (named Anthrax Toxin Receptor for the way it was discovered), which is found on many cell types. This has allowed medical researchers to deliver proteins broadly to all cells, but not target delivery to specific cell types.
To develop a cell-specific delivery platform, Chen et. al. concentrated on the enterotoxin LTIIa, originally identified in E. coli. LTIIa is an AB5 toxin, meaning five B subunit proteins interact with a single A subunit protein. LTIIa binds the ganglioside GD1b, a cell receptor enriched in nervous system cells.
Its A subunit has two domains: A2, which interacts with the five B subunits, and A1, which contains the toxic enzyme activity (see the schematic, taken from Fig 1). These two subunits are connected by a disulfide bond, which is cleaved after entry into the cell. The authors’ clever hypothesis was that the A1 domain could be engineered to a non-toxic protein – maybe even a therapeutic.
To test this idea, they first substituted β-lactamase as the protein associated with A2. Using this engineered system, β-lactamase was efficiently delivered to cell cytoplasms, where the β-lactamase separated from the A2 domain after entry to the cell. The protein was active once delivered, and its activity was dependent on the amount of protein delivered. The fact that the β-lactamase separated from the rest of the delivery system is important when considering cargo may require particular localization after delivery for proper activity.
Because all AB5 toxins interact with various gangliosides (membrane glycosphingolipids with various sialic acid modifications), the authors took care to show specificity for neuron delivery. While other related gangliosides, such as GM1a, did allow some non-specific delivery, delivery was at least twice as specific in GD1b-expressing cells, and was highest of all in neurons.
In the final proof-of-principle experiment, the scientists used their system to deliver a therapeutic to poisoned neurons. Neurons were exposed to botulinum neurotoxin A (BoNT/A), which prevents synaptic vesicle fusion by cleaving SNAP25. The authors used an engineered LTIIa variant that carried an inhibitory antibody against this toxin. When the cells were treated with increasing concentrations of the LTIIa-antibody, there was greater inhibition of cleaved SNAP25. This inhibition didn’t work on a second type of neurotoxin (BoNT/D), demonstrating the specific effect of the delivered antibody protein.
What are the implications for such research? One major advantage of protein delivery over gene delivery is the lack of genetic component, allowing for fewer nonspecific effects involving DNA integration. This also facilitates dosed and short-term treatments. Between latent viral infections, neuronal tumors and fibromas, and Alzheimer’s and other neuro-degenerative diseases, the array of possible diseases benefiting from this delivery platform is broad!
Targeted drug delivery facilitates a higher concentration delivered with fewer side effects. While the authors here tested a protein cargo, the delivery platform may eventually be broadened to deliver non-protein cargo. This is a great step forward toward engineering a microbial toxin delivery system for human therapeutic use!
Image credit: AB-toxin schematic, Figures from Chen et. al.
Do you enjoy lox on your bagel? A refreshing ceviche in the hot summer weather? New research published this week in Applied and Environmental Microbiology highlights the importance of proper storage of salmon and other meats that require no cooking prior to eating.
The research, published by Silin Tang et al out of Dr. Teresa Bergholz’s lab, focused on cold-smoked salmon as a model of ready-to-eat seafood. During its preparation, cold-smoked salmon isn’t heated to a temperature high enough to kill microbes, leaving it vulnerable to Listeria monocytogenes growth. Exposure of meat to L. monocytogenes is particularly dangerous, since this bacterium can grow even at refrigeration temperatures (ideally 1.6°C, or 35°F – minimally below 40°F).
The scientists wanted to examine what genes L. monocytogenes turns on and off when growing on salmon compared to a laboratory medium with similar salt, pH, and temperature conditions. Many experiments have been run looking at L. monocytogenes gene expression, but most of these have been conducted using laboratory medium – a great way to identify genes used in a single variable but not reflective of the environment the bacteria will actually experience. Experiments that have looked at expression on different food sources have had to deal with different growth rates on foodstuffs versus lab medium.
Fortunately, L. monocytogenes grew similarly well on salmon and in traditional lab medium. This meant the experimental time points would yield a similar number of bacteria, and possible confounding factors would be minimized. Using RNA-seq, the scientists found about 150 genes diffentially expressed in salmon-grown Listeria: 88 with increased expression and 61 with decreased expression, relative to growth on lab medium. What kinds of genes?
One standout group of genes was not metabolically-linked: a set of five genes known to be regulated by a virulence regulator, PrfA. One of these genes, a phospholipase, may digest salmon cell membranes, making available lipid components like ethanolamine for bacterial growth through the upregulated metabolic pathways.
Listeria infections are a serious cause of foodborne illness (1600 hospitalizations and 260 deaths annually in the US) – you may remember several recalls related to Listeria contamination in the past year. Fear of these outbreaks is one reason New York now requires all fish to be frozen before serving. The research from Tang et. al. aims to understand how bacteria grow on foodstuffs like cold-smoked salmon. Applications from understanding bacterial metabolics on salmon may generate better precautionary measures in food preparation or be useful in identifying contaminated items before outbreaks begin!

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