Exam 2 Overview

2.1 Genomics and infectious disease

a) Population genome structure, evolution, and genomic epidemiology

• Selective pressures act on gene diversity: net positive or negative

• Vertically and horizontally inherited DNAs have different evolutionary histories: We care about vertical when determining evolutionary history

b) A genomic epidemiology analysis of a devastating cholera outbreak

• The Haitian V. cholerae isolates were essentially identical, consistent with them being
  clonally-related. Meaning the outbreak was cause by a single strain.

• Genomic data supported the conclusion that the Haitian outbreak was caused by the introduction of a pathogenic Nepal strain from UN peacekeepers because the 2 had essentially identical core genomes (mobile genetic elements don’t matter).

c) The four virulence-related themes revealed by analysis of microbial genomes

• Many virulence genes are encoded by mobile gene elements, so horizontal gene transfer play a major role in acquisition of virulence genes.

  • Eg: Not all S. aureus strains have gene that encodes exfoliating toxin, so they can’t all cause scaled skin syndrome.

• Virulence genes aren’t limited to pathogens

  • Eg: Legionella virulence factor IcmT facilitates escape of the bacterium from
    human macrophages, but also from the far more ancient predator the free-living amoeba

  • Eg: Gut symbionts in humans and livestock are exposed to antibiotic therapies, so our guts are a natural reservoir for resistance genes as they are selected for.

• There is diversity in the number of genes associated with virulence.

  • A lot of times species with highly repetitive DNA have higher mutation rates due to repeat driven genome expansion and recombination.

  • Also there is diverity within the virulence genes themselves. Like variable surface proteins to evade immune detection/recognition.

• Genome reduction and pseudogene formation is common in pathogens

  • Host adapted strains have less stringent selection for genetic variety, so they are more likely to form pseudogenes and have genome reduction to get rid of metabolic burdens and take advantage of deleterious mutations compared to generalist strains.

d) Antibiotic resistance and antibiotic treatment plans

• Many resistance genes are coded by a single protein, so they can be discovered easily by sequencing. But, using this method does not always accurately determine resistance because there can be mutations or new resistance genes that are missed.

e) Genomic surveillance of human pathogens

• Capsular switching: a serotype targeted by the vaccine with switch capsule gene with a non targeted serotype to escape vaccine coverage. This switching is caused by the strong selective pressure exerted by vaccines and can lead to vaccine inefficacy.

2.2 GAS genomics

• The group A Streptococcus (GAS; a.k.a. Streptococcus pyogenes)

  • disease manifestations

    • Direct infection: GAS pharyngitis (strep throat), puerperal infections, necrotizing fasciitis, TSS, Pyoderma

    • Post GAS infection: Pediatric autoimmune neuropsychiatric
      disorders (PANDAs), Acute rheumatic fever (arthritis/carditis), Acute post-streptococcal glomerulonephritis (APSGN)

  • serotyping: Based on classical GAS virulence factor, called M protein. It induces type specific immunity (only to same serotype). Candidate target for vaccine, but serotype switching will occur and make vaccine ineffective.

  • genome: Often poly lysogenic with lots of variation in phage presence/absence. Usually essential and highly expressed genes are on leading strand (gene strand bias)

• Insights gained because of GAS genomics:

  • re-emergence of GAS

    • Horizontal gene transfer and the 1980s re-emergence: Used phylogenetic analysis to figure out distinguishing factors between old and new GAS. Figured out a series of molecular changes created a contemporary M1 clone. SNPs are not uniform due to selective pressures. So, regulator-encoding genes are often“hot spots” for mutation

    • Gene-specific variation frequency: Also determined how to use molecular clock ( correlation between increasing genetic distance and increasing year of isolation) to determine genetic distance

  • molecular basis of serotype-specific phenotypic variation

    • The association of M3 GAS with cases of severe invasive infection: Figured out that wile gene content was largely similar, different isolates can expressed different virulence profiles from the same genes due to regulatory systems. Only difference is mutated RocA

      • Used a mouse model to figure this out including parent M3, derivative with fixed rocA (controlled that any differential expression we see is directly due to mutant rocA function), and then derivative of fixed rocA where it is not re-mutated (controlled for potential unintended changes that might have occurred during the initial correction)

      • The association of M28 GAS with cases of puerperal sepsis: Caused by gain of RD2 virulent genomic island acquired from GBS by horizontal gene transfer.

2.3 Yersinia Pestis

• The Yersinia pathogens:

  • 14 species clusters- includes pathogenic and nonpathogenic

  • Used as a model to understand pathogenic evolution because we have examples of both long and short term evolution.

  • Y. enterocolitica and Y. pseudotuberculosis: Zoonotic pathogen that cause self limiting gastroenteritis (enteropathogens)

  • Y pestis: Zoonotic agent that causes the plague as it is transmitted from fleas/rodents to humans (nonenteropathogen)

  • Yersinia pathogens are present in 2 distinct linages from distinct gene loss and gain events

  • Y. pestis evolved from Y. pseudotuberculosis through gene acquisition and loss.

  • Foothold moments during evolution of pathogenic Yersinia

    • acquisition of the pYV virulence plasmid, which encodes the Ysc type III secretion system (T3SS)

• The epidemiological and clinical features of Y. pestis

  • Historical importance:

    • 1st pandemic: Justinian plague killed 25-50% of europe

    • 2nd pandemic: Black death killed 25% of the world

  • Epidemiology: Transmitted usually by flea bites, but also direct contact and person to person. Cat A biothreat

  • Clinical:

    • Bubonic plague: Infection following flea bite causing painful bubos (high mortality untreated)

    • Pneumonic plague: Infection following aerosol exposure leading to severe respiratory symptoms and high mortality if untreated

    • Septicemic plague: usually follows bubonic infection that spreads to the bloodstream, causing sepsis and potentially leading to multi-organ failure

• The evolution of Y. pestis from Y. pseudotuberculosis

  • Y. pestis lost ability to colonize intestine: 150 required genes became pseudogenes due to high levels of insertion sequences from frequent genome rearrangements.

  • Y. pestis gained flea foregut colonization: has a pFra plasmid which encodes ymt. Ymt promotes flea borne transmission (in fleas fed with mouse, human or rat blood), but not virulence. We know this because Y. pestis that are ymt- can still cause disease in humans, they just can’t infect as many vectors

  • Y. pestis enhanced biofilm abilities: Biofilms require hemin storage (hms) locus. Hms transcription is activated by c-di-GMP. But c-di-GMP is repressed by PDEs and hms is repressed by rcsA repressor proteins. Y. pestis have mutated PDEs and rcsA that decrease their activity so that hms is not repressed.

  • Y. pestis lost oral transmission abilities: Urease is required for oral transmission, but urease is toxic to fleas. Y. pestis has ureD mutation that takes away urease so they can still thrive in fleas.

  • Key Y. pestis virulence factor: pPla plasmid contains plasminogen activator (Pla), which allows bacteria to disseminate from flea bite location to lymphatics and cause buboes. buboes most often appear at groin first because it is the closest lymph node to lower extremities where most flea bites occur.

• What paleomicrobiology is (and how the study of Y. pestis has contributed to it)

  • microbiological study of prehistoric material and, in part due to well-kept burial records of plague victims, genomic studies of Y. pestis have been major contributors to this field

  • DNA is damaged overtime, including deamination (deamination of cytosine to uracil)

  • Ancient DNA genomics were analyzed first using DNA capture arrays which enriched DNA to search for Y. pestis presence in Black death and Justinian plague victims. But later a metagenomic sequencing approach was used, which was better because it allowed them to look at the genome as a whole and find that not all bronze age Y. pestis had the Ymt toxin yet.

• Phylogenetics of pandemic Y. pestis

  • Subdivided into 5 different biovars (based on sugar fermentation and nitrate reduction)

  • Strains associated with the 1st (Justinian Pandemic): Phylogenetically distinct from 2nd and 3rd plague, and appear to have no living descendants

  • The strains associated with the 2nd (Black death pandemic): Phylogenetic position suggests that this strain gave rise to ALL LATER Y. PESTIS STRAINS. This one spread from Asia to Europe, then later to china where it emerged as the 3rd pandemic.

  • Strains associated with the 3rd pandemic: All caused by 1.ORI group, which started in China, then to Hong Kong, then globally including US (introduced on Hong Kong Ships).

  • all Y. pestis isolates in the United States are descendants of 1.ORI1.d

2.4 Mycobacteria

• The mycobacteria:

  • The M. tuberculosis complex (MTBC): genetically-related group of species that can cause tuberculosis in humans or other animals

  • Tuberculosis: Low infectious dose; Most people have asymptomatic primary infection, because it remain dormant as a latent infection, then years later it reactivates.

  • Epidemiology: Humans are only reservoir. WHO thinks 1/3 world has latent. TB in US has declined, its mostly in IS, homeless, and addicts

• Genomic insights into mycobacteria evolution:

  • Mtb has highly repetitive DNA

  • You can tell members of MTBC apart using presence or absence of certain regions of difference (RDs) that represent markers of unidirectional deletion events, and by
    certain SNP

• The Bacille Calmette-Guérin (BCG) vaccine: live attenuated vaccine that, since 1921, has been used to protect against Mtb infection

  • The BCG vaccine was created by Calmette & Guérin who passaged an M. bovis isolate 230 times, to help it lose virulence while retaining immunogenicity. But over-passage has caused varied T-cell epitopes in BCG strains, leading to differences in vaccine efficacy and immune response among different populations. To see which epitopes were lost during attenuation, look for RDs present in all of the 13 BCG strains. Any that happened after will have variable RDs in BCG strains.

• Methods for genotyping M. tuberculosis isolates

  • Spoligotyping-Looking for presence or absence of 43 spacers in CRISP region. Read using 15-digit code, first position is 4, 2nd is 2, and last is 1. So, 111 would be 7. 100 would be 4. 110 would be 6.

  • MIRU-VTNR- analysis of DNA segments containing tandem repeated
    sequences in which the number of copies of the repeated sequence varies among strains

  • IS6110-RFLP - detects variations in the number and location of IS6110 elements in the genome

  • Whole Genome Sequencing (WGS) - provides comprehensive data on the entire genome of the isolate, allowing for detailed phylogenetic analysis and identification of mutations.

  • Which do we want to use most- WGS has most discriminatory power and can be used to trace outbreak transmission chains.

• Tuberculosis in the Americas pre-Columbus:

  • Data indicates the seals carried M. pinnipedii from Europe of America, pre-1942. Then this strain was replaced by a European strain, post contact (1942)

S. aureus:

• Adaptive mutations and selective sweeps

  • AM: advantageous mutations

    • Can become fixe din a population rapidly through selective sweeps

  • Transmission bottlenecks: Reduce population diversity because only a subset

  • Genome selective sweeps: when a strain with AM outcompetes other strains in a population

  • Gene selective sweeps: When a gene harboring an AM is shared from one strain to the other strain in a population

    • Differences: both have different effect in genome level variation in a population. Genome selective will make it so that the entire population has the SAME GENOME. Gene selective means all isolates in the pop will have that GENE, but some genome variation.

• Introduction to Staphylococcus aureus

  • Overview of diseases:

    • Can cause a range of infections including skin/soft tissue infections. Also toxin associated disease like TSS, food poisoning, and SSS.

  • Mobile genetic element-encoded toxins:

    • In S. aureus the mobile genetic elements are mobilized by helper phages.

    • Exfoliating toxin: Encoded within mobile genetic elements!

    • SaPI1: Codes for toxins like TSST-1 and SSEQ and SEK

    • SaPI3: COdes for SEB, SEQ, and SEK

    • Why can’t strains have both pathogenicity islands? Each island shares the same integrase gene, which suggests that they cannot coexist within the same bacterial strain due to the competition for integration sites.

• Emergence of antibiotic resistance

  • Methicillin-resistant S. aureus (MRSA)

    • Methicillin resistance comes from MecA which encodes a penicillin binding protein. MecA lies within a mobile element called the staphylococcal chromosome cassette (SCCmec).

    • It is hypothesized that mecA appeared before human antibiotic use. So it was actually from selective pressures with hedgehog infections because they naturally have beta lactam antibiotics.

  • VISA & VRSA- Vancomycin targets peptidoglycan synthesis

    • VISA: Have reduced sensitivity (But can be treated with a higher concentration). Due to mutations in specific genes (NOT HGT) which cause a thicker cell wall.

    • VRSA: Are resistant to vancomycin because through HGT they got the vanA operon. The operon changes that instead of
      terminating in D-Ala-D-Ala they terminate in D-Ala-D-lactate, to which vancomycin does not bind. This likely came from a HGT of a transposon containing vanA operon from a resistant enterococcus.

• Animal reservoirs

  • Companion and livestock animals:

    • Livestock animals are more likely to have animal adapted MRSA strains (higher likelihood of severe infections in animals).

    • Companion animals are more likely to have MRSA strains that are similar to human MRSA clones

  • Genetic adaptation

    • S. aureus is mostly a human pathogen because their cell receptors have a higher affinity for human proteins.

    • Livestock S. aureus isolates lose factors that only function in human pathogenesis via genetic adaptation.

      • Ex: SpA bind to human IgG to inhibit antibodies. Avian staph does not have SpA expression because it doesn’t work for IgY (avian IgG analog)

• Emergence of the hyper-virulent USA300 S. aureus lineage

  • Novel mobile elements

    • Phage φSA2usa- codes for Panton-Valentine leucocidin (PVL),
      a pore-forming toxin that induces neutrophil death

      • role in hypervirulence is controversial because at first a study said it directly related to hemolytic activity, but this was disproved.

    • Arginine catabolic mobile genetic element (ACME)

      • Codes for speG gene which allows bacteria to survive in presence of polyamines spermidine and spermine.

      • Likely obtained through HGT from S. epidermidis