Overview

Bovine mastitis is the most costly agricultural disease, plaguing dairy cattle worldwide. It costs the United States around $2 billion annually, half of which is due to subclinical mastitis [1]. Traditional treatments usually involve the use of antibiotics. However, the use of antibiotics has led to increasing concerns of resistance as well as negative implications on human health. Furthermore, the development and synthesis of new antibiotics is slow and may not necessarily have the desired effects.

Bacteriocins are natural antimicrobial peptides secreted by bacteria to gain competitive advantage in their natural ecosystems. Functionally, bacteriocins kill target cells via different mechanisms, including pore formation, inhibition of translation, inhibition of transcription, and inhibition of cell wall synthesis. The use of bacteriocins as antimicrobial drugs has three significant advantages. First, bacteriocins are known to not be harmful to humans. One such group of bacteriocins, the Nisin family, is approved by the Food and Drug Administration as a food preservative [2]. Second, bacteriocins have a remarkably fast evolution rate [3]. Finally, bacteriocins are known to be very specific to certain species within the same genus, although some are known to have broad spectrum activity as well. This diversity among bacteriocins, along with their safety for human consumption, makes them a promising alternative to traditional antibiotics.

The pathogens of bovine mastitis can be divided broadly into Gram positive and Gram negative species, as well as contagious or environmental species. Our choice of bacteriocins is such that we can target the pathogens from all categories. Some potent bacteriocins have incredibly complex post-translational modifications, but we have also chosen ones that don’t require any modifications. The genes will be cloned into the standard pSB1C3 vector, and the peptides heterologously expressed by inducing the expression of T7 RNA Polymerase in Escherichia coli BL21(DE3). The efficacy of these recombinant proteins will be tested via zone of inhibition assays and growth curve assays.

Chassis

BL21(DE3): BL21(DE3) contains the bacteriophage T7 RNA polymerase gene under the lacUV5 promoter and is deficient in common proteases. Therefore, this strain optimizes the expression of proteins placed under a T7 promoter after IPTG induction [5].

XL1B: XL1-Blue supercompetent cells allow for blue-white screening of recombinant plasmids. In addition, XL1B cells are endonuclease and recombination deficient, which greatly improves miniprep DNA quality and yield [6].

Mastitis-Causing Pathogens

Bovine mastitis is caused by a diverse group of pathogens, ranging from bacteria to algae. Here, we list some of the bacteria that are known to be the most historically significant pathogens, the most virulent pathogens, as well as the most common pathogens.

Enterobacter spp.: Enterobacter spp. are mastitis-causing, Gram-negative bacteria. They are found in the environment, specifically areas such as manure, bedding, soil, and muddy lots or corrals. Enterobacter spp. spreads to a cow’s mammary gland through environmental contact, especially when teats come in contact with manure or contaminated bedding. Notably, these bacteria do not respond well to antibiotic therapy. Therefore, it is very important to keep cows clean and dry and employ proper milking procedures to prevent infection from Enterobacter spp [7].

Enterococcus spp.: Enterococci spp. are Gram-positive bacteria and common environmental pathogens of bovine mastitis, contributing to 11% of cases in Central California. It has been shown to be resistant to a wide range of antimicrobial compounds [8].

Escherichia coli: E. coli is an environmental pathogen and a very common cause of mastitis in dairy cattle, with severity ranging from subclinical infections to severe systemic diseases. Antimicrobial treatment options are very limited and with negative implications on human health [9].

Klebsiella spp.: Klebsiella spp. is a group of environmental pathogens that are significant causes of bovine mastitis, with rising incidence rates in recent years. Klebsiella species are known to stay dormant in the udders and spread through cattle quickly. Infected cattle often die within days, and evidence suggests increased antibiotic resistance [10].

Lactococcus lactis: Lactococcus lactis is a Gram-positive bacteria. It was not identified as a mastitis-causing pathogen until the start of 200,7 when five New York state dairy herds were infected. It is often hard to identify in laboratories, and as a result is often misidentified as Streptococcus or Enterococcus. The treatment of these organisms is difficult because they have the ability to transfer genetic material of resistance to commonly used antibiotics. These bacteria can be spread from cow to cow through milking, but most often, it is spread environmentally, such as through bedding [11].

Pseudomonas spp: Pseudomonas spp. is a genus of bacteria that are mastitis-causing pathogens. Its infection rate in cows is around 1-3%. Using water supplies of all kinds, such as parlor water hoses, or using contaminated teat dips and contaminated infusion equipment for cows are all sources of Pseudomonas spp. infections. This genus of bacteria often attacks weak or injured tissues in the teats or mammary glands, and it can cause non-clinical cases of bovine mastitis, but exposure to large numbers of it can lead to an outbreak of clinical cases [12].

Streptococcus agalactiae: Streptococcus agalactiae is a Gram-positive bacteria that infects the bovine mammary gland. Presence of S. agalactiae is associated with increased somatic cell count and decreased quality of milk. It is present in 11% to 47% of cow herds [13].

Staphylococcus aureus: Staphylococcus aureus, a Gram-positive coccal bacteria, and is one of the most devastating mastitis causing pathogens. This is mainly because of its ability to form biofilm. As a result, S. aureus is very difficult to treat with antibiotics as drugs are often rendered ineffective due to transport mechanisms, even if effective at killing the cells. Thus, it is one of the worst pathogens for dairy farmers [14].

Streptococcus uberis: Streptococcus uberis is Gram-positive and is the most common Streptococcus species that causes bovine mastitis. S. uberis are commonly found in manure, bedding, and other organic matter. Therefore, it often spreads to uninfected cows through environmental contact. S. uberis can also spread from cow to cow during milking [15].

Parts Origin

Bacteriocins are incredibly diverse and have very specific activity against target species. Below we list some of the species that produce bacteriocins that are known to target the pathogens that cause bovine mastitis. We optimized the gene codons for overexpression in E.coli.

Bacillus subtilis: Bacillus subtilis is a Gram-positive endospore forming bacteria. It is able to withstand high temperatures and dry environments, and it is commonly found in soil, air, or plant compost. When B. subtilis is active, it produces many enzymes. Research indicates that it can easily be genetically manipulated, and that it can be used as a broad spectrum antibiotic [16].

Klebsiella pneumoniae: Klebsiella pneumoniae are rod-shaped Gram-negative bacteria that are ubiquitous in nature. They are known to colonize in gastrointestinal tracts and form large polysaccharide capsules to prevent phagocytosis. They are especially common in farms around New York and are gaining antibiotic resistance. K. pneumoniae and several species are known to be opportunistic pathogens [17, 25].

Lactobacillus salivarius: Lactobacillus salivarius is found in human, porcine, and avian gastrointestinal tracts. L. salivarius is a probiotic species because it exhibits probiotic properties such as the ability to modulate gut microbiota, produce antimicrobial substances (namely, bacteriocins), and stimulate protective immune response, among others [18].

Lactococcus lactis: Lactococcus lactis is informally known as Lactic Acid Bacterium because it ferments lactose to lactic acid. It is Gram-positive and the producer of a few of the bacteriocins we worked with, including Nisin F, Nisin Z, and Nisin A. This bacteria is found to be associated with plants, mainly grasses, and from there, can be incorporated into milk [19].

Staphylococcus aureus: Staphylococcus aureus is a Gram-positive coccal bacteria, commonly found on the skin and in the respiratory tract of humans. It is non-pathogenic in some cases, but can often result in infections of the skin, or food poisoning in humans. It produces the bacteriocin Aureocin, which can be used to treat various staphylococcal mastitis causing pathogens. It attacks these strains by facilitating generalized membrane destruction, killing the bacteria [20, 21].

Staphylococcus epidermidis: Staphylococcus epidermidis is a Gram-positive bacteria that is part of the normal human flora. Its hosts often include humans and other warm-blooded animals, and many strains of the species can survive on dry surfaces over a long duration of time. This bacteria secretes a slime-like substance that allows it to adhere to plastic and to cells, and its infections are often linked to intravascular devices, such as prosthetic heart valves, as well as to catheters and large wounds [22].

Staphylococcus simulans: Staphylococcus simulans is a Gram-positive coccus known to colonize mammalian hosts. It is best known for its ability to produce lysostaphin, a metalloendopeptidase known to be extremely effective against Staphylococcus aureus [23].

Streptococcus uberis: Streptococcus uberis is a Gram-positive bacterium and the producer of one of the bacteriocins we worked with, Nisin U. Nisin U is a lantibiotic and acts by forming pores in target cells. Nisin U is active against various other Streptococcus species, and some Staphylococcus species [24].

Results

We first show that there is regulated expression of the bacteriocins in E. coli.

Western blot reveals regulated expression of bacteriocins. In the image on the left, expression of Bactofencin A (BBa_K1931006), Enterocin E760 (BBa_K1931007), and truncated MBP (BBa_K1931005) were observed (lanes 3,4,5) whereas no signals were detected from uninduced negative control (lane 2). On the right, expression of lysostaphin (BBa_K1931011) was observed in the crude extract and the eluent fraction (evident from the two significant signals).

We proceed to characterize the efficacy of the bacteriocins via zone of inhibition and growth curve assays:

Zone of inhibition data for Staphylococcus aureus and Staphylococcus epidermidis. Purified maltose binding protein (MBP) was used as a negative control, and the beta-lactam antibiotic ampicillin was used at standard working concentration (100 ug/mL). Total protein concentrations for bacteriocin samples were adjusted to 1 mg/mL. Error bars denote a 95% confidence interval. BBa_K1931006 and BBa_K1931011 displays promising efficacy against S. aureus. BBa_K1931007 displays promising efficacy against S. epidermidis.

Zone of inhibition data for several species of gram negative bacteria known to cause mastitis in dairy cattle. Purified maltose binding protein (MBP) was used as a negative control, and the aminoglycoside antibiotic kanamycin was used at standard working concentration (30 ug/mL). Total protein concentrations for bacteriocin samples were adjusted to 1 mg/mL. Citrobacter freundii is not a significant pathogen of bovine mastitis, but is a known target of Enterocin E760 (BBa_K1931007). Error bars denote a 95% confidence interval. All experimental samples display efficacy not significantly different from that of kanamycin.

Based on the success of Bactofencin A (BBa_K1931006) in our zone of inhibition analysis, we tested its efficacy over time with a growth curve analysis. Ampicillin was added to achieve standard working concentration (100 ug/mL). An equivalent volume of protein sample was added to the culture. MBP was used as a negative control. In the span of four hours, Bactofencin A displayed similar levels of efficacy as ampicillin.

A similar growth curve assay was performed for S. epidermidis with BBa_K1931007. Here, the bacteriocin again demonstrates similar efficacy to antibiotics.

A growth curve was constructed for E. coli, but this time with kanamycin as the antibiotic of choice. The expression of Colicin M (BBa_K1931009) was induced with IPTG, and OD600 was monitored over time. Again, Colicin M demonstrates similar levels of efficacy as kanamycin.

A growth curve for C. freundii was constructed. BBa_K1931007 displays similar levels of efficacy as that of kanamycin.

Future Works

Despite their small sizes, bacteriocins are known to be extremely diverse with complex post-translational modifications. This presents a challenge to us as we attempt to purify them and to characterize their activities.

Nisins and Subtilins undergo extensive post-translational modifications. Well-conserved serines are dehydrated to form lanthionines, which then undergo intramolecular Michael additions. Finally, leader sequences are cleaved to yield mature antimicrobial peptides, and a transport protein delivers the bacteriocin to the extracellular environment. As a first step, Cornell iGEM hopes to characterize these proteins by co-expressing the enzymes that catalyze dehydration and cycloaddition, then purify these peptides in large quantities. An enterokinase cleavage sequence will be used to cleave the leader sequence to yield the mature bacteriocin.

The Nisin family of bacteriocins undergo complex post-translational modifications that involve dehydration and intramolecular Michael additions, followed by cleavage of a leader sequence. The result is a complex penta-ring structure that is resistant to protease cleavage [26].

One difficulty we faced in protein purification can be attributed to the vastly different properties of these peptides, which led to low protein yields. Furthermore, the only resin available to us was HisPur Cobalt Resin from Thermo Fisher Scientific. In the future, it would be ideal to experiment with different columns and resins to determine the optimal protocol for purification.

In addition to zone of inhibition assays and growth curve analyses, we would like to have more quantitative information regarding the potency of bacteriocins. In the future, we hope to test these bacteriocins using dose-response curves to determine EC50 and the minimum inhibitory concentration.

Another important consideration is the shelf life of these bacteriocins compared to antibiotics, especially because proteins tend to be relatively unstable outside of native environments. We would like to test the relative stabilities of these peptides at different temperatures, and compare this to the antibiotics typically used to treat bovine mastitis.