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Advanced Medical Microbiology > Progress Exam 4 (November 12 - 13 - 14 - 15) > Flashcards

Progress Exam 4 (November 12 - 13 - 14 - 15) Flashcards

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1
Q

Incubation time of Coxiella burnetii

A

2 - 3 weeks

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2
Q

Percentage of people developing Q-fever after infection with C. burnetii

A

40%

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3
Q

Characteristics of Coxiella burnetii

A
  • Gram-negative: obligate intracellular
  • Q-fever (acute or chronic) - zoonosis: aerosolized soil or animal products
  • Tick transmitted
  • Cattle-sheep-goats (wild animals; arthropods)
  • Infection: high morbidity
  • Bacterium is stable: category B select agent
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4
Q

What is the tropism for professional phagocytes?

(Tropism is the ability of a given pathogen to infect a specific location)

A

Enter via receptor mediated endocytosis

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5
Q

What is the phagolysosome-like compartment of Coxiella?

A

Coxiella-containing vacuole: CCV

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6
Q

Distinct developmental stages of C. burnetii:

A
  • Small cell variants (SCVs): metabolic inactive, resistant and extracellular
  • Large cell variants (LCVs): metabolic active, intracellular (acidification triggers transcription)
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7
Q

What are the target cells for C. burnetti?

A

Alveolar macrophages

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8
Q

How does C. burnetii enters the alveolar macrophages?

A

Passively entering via actin-dependent phagocytosis (alpha-v-beta-3-integrins). That normally activate the immune system, but not with C. burnetii –> silent infection.

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9
Q

Host cell receptor alpha-v-beta-3 integrin:

A
  • Involved in removal of apoptotic cells via phagocytosis
  • Inhibition of inflammation: silent infection
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10
Q

Infection of macrophages by C. burnetii

A
  1. Bind to actin filaments
  2. Nascent CCV (effector proteins already present; merging with autophagosome) –> pH 5.4
  3. Maturing CCV (merging with lysosome) –> pH 5
  4. CCV with a pH of 4.5 –> bacteria can perfectly grow
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11
Q

From phagosome to CCV

A
  • Early phagosome: acquires small GTPase RAB5
  • Stimulates fusion with early endosome –> acidification pH 5.4
  • Acquires EEA1 (early endosomal marker 1)
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12
Q

What is the function of RAB5?

A

It stimulates fusion with early endosome

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13
Q

Characteristic of late phagosome (in CCV cycle)

A

Lacks RAB5 but contains GTPase RAB7 and:
* LAMP1 - Lysosome Associated Membrane Protein
* ATPase - proton pump pH 5

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14
Q

Characteristics of mature CCV

A
  • Heavily loaded with bacteria: large CV –> small CV
  • Retains capacity to fuse to expand
  • Same markers as the early, but now also anti-apoptotic markers: BCL2, BECN1 and Erk1) –> prevent release from cytochrome c from mitochondria
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15
Q

Key hallmark of Coxiella

A

Really close in contact with the membrane of the host cell

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16
Q

In the membrane of Coxiella burnetii there is a …

A

Type IV secretion system (T4SS)

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17
Q

What is the name of the type IV SS in C. burnetii?

A

Defect in organelle trafficking (Dot) / Intracellular multiplication (Icm)

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18
Q

The Dot or Icm secretion system

A
  • Is encoded on islands with relatively low GC contents
  • Eukaryotic protein motifs
  • Secretion of effector proteins (DotF and DotG)
  • Polymorphic regions between different pathotypes
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19
Q

Time of expression T4SS in C. burnetii

A

After 8 hours and expression requires low pH

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20
Q

What is an important response regulator in the Dot/Icm type IV secretion system?

A

PmrAB (regulates transcription; low pH stimulus)

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21
Q

Fur-transcriptional regulator

A
  • Excess iron –> binds to Fur and complex binds to Fur-box –> blocking RNApol binding –> no transcription
  • Low iron –> RNApol can bind –> transcription
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22
Q

Effect of knock-out PmrAB

A

Impaired growth of C. burnetii

Conclusion: needed for growth

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23
Q

What did the luciferase transcriptional reporter system proved about the PmrA box sequence?

A

Knocking out the regulator –> no expression or less expression of PprmA

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24
Q

PmrA binds to the PmrA consensus box and … transcription

A

Stimulates

In contrast to Fur

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25
Q

PmrA model

A
  • High pH –> non-active PmrA –> no transcription (is the promotor region blocked?
  • Low pH –> active PmrA –> active transcription –> effector proteins (Icm/Dot): modification vacuole
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26
Q

Fur is a repressor protein and uses:

A

Repression by steric hindrance

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27
Q

PrmA is an activator protein and uses:

A

Class I activation
Class II activation
or
Activation by conformation change

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28
Q

Proteins domains (signature) point function

A
  • Ankyrin repeats
  • Coiled-coil domains
  • Tetratricopeptide repeats
  • F box-domains
  • Fic domains
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29
Q

Pathogens from poultry

A

Salmonella, Campylobacter, VRE, ESBL + E. coli, H7N7 and other avian influenzas

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30
Q

Pathogens from pigs

A

MRSA and hepatitis E virus genotype 3

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31
Q

Pathogens in cattle

A

Bovine Spongiform Encephalopathy –> vCJD

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32
Q

Pathogens in goats

A

Q-fever

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33
Q

Symptoms before GP in Herpen knew it was Q-fever?

A
  • Respiratory infections
  • Atypical pneumonias
  • Hospital admissions of his patients
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34
Q

Q-fever in humans:

A
  • 60% asymptomatic
  • 20% flu-like (head ache, fever, nausea, muscle pain)
  • 20% serious (persisting fever, chest pain, severe head ache, diarrhoea, vomiting, often: atypical pneumonia
  • 1 - 3% chronic infection: endocarditis and other intravesicular infections.
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35
Q

Antibody detection against C. burnetii

A
  • IgG-phase 2 antibodies in the acute setting
  • IgG-phase 1 antibodies in chronic Q-fever
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36
Q

Treatment of acute Q-fever

A

Doxycycline

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37
Q

Treatment of chronic Q-fever

A

Combination therapy

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38
Q

What did the government do to get rid of the 2009 Q-fever outbreak?

A
  1. Culling of all infected herds.
  2. Vaccination of all healthy goats.
  3. Ban on all goat transports.
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39
Q

What percentage of CT content as a cut-off for PCR?

A

40% CT content

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40
Q

Conclusion/recommendation of Hans Zaaijer’s lecture

A

Agricultural affairs and economics should not have been allowed to prevail over public health.

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41
Q

Pre-exisiting infection and antibodies need to be ruled out for … before vaccination.

A

Mycoplasma infections
C. burnetii infections

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42
Q

Function of villi and microvilli

A

Maximize the absorptional capacity

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43
Q

Specialized structure in the bottom of the villi

A

Crypt with Paneth cells, CBC cells (stem cells)

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44
Q

Butyrate

A

Toxic to stem cells, but energy source for epithelial cells

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45
Q

Gut microbiota contribute to …

A
  • Dietary compounds
  • Vitamins
  • Fiber (short chain fatty acids)
  • Immunity training
  • Inflammation regulation
  • Pathogen resistance
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46
Q

Transplantation of gut microbiota of obese mouse to germ free mice

A

Increase in bodyfat after the transplantation

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47
Q

Single bacterium effect after transplantation

A

Already increases the vascular network comparable to the vascular network after whole microbiota transplantation

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48
Q

New way of treating Clostridium difficile

A

Fecal microbial transplantation (FMT)

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49
Q

Small intestine vs. large intestine

A
  • Small intestine villi and crypts
  • Large intestines only crypts
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50
Q

Gut-Microbe (GuMi) physiomimetic system

A

Microbiota, mucus layer and colon epithelium.

Recirculation of oxygen-rich media

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51
Q

Marker of the goblet cells shows …

A

Muc2 shows that the GuMi maintains intact monolayers and multiple cell types.

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52
Q

What is the effect of F. prasnitzii in the microbiota?

A

Changed cytokine/chemokine profile and gene expression in the presence of CD4+ T cells.

Changes immune responses and calm down colon epithelium.

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53
Q

Biomaterial-associated infections (BAI) mostly caused by

A

Staphylococcus aureus

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54
Q

Anti-microbial coating designs

A
  1. Antimicrobial-releasing coatings
  2. Contact-killing surfaces
  3. Nonadhesive surfaces
  4. Ideal multifunctional coatings
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55
Q

Examples of anti-adhesive antimicrobial coatings

A
  • Reduced adherence of bacteria: hydrophilic surface
  • Reduced adherence of plasma proteins: polymeric phospholipids/polyethylene glycol PEG
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56
Q

Examples of anticoagulant/microbicidal antimicrobial coatings

A
  • Benzalkonium chloride heparin
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57
Q

Examples of microbicidal antimicrobial coatings

A
  • Antiseptics: chlorhexidine-silver sulfadiazine
  • Metals: silver, silveroxide
  • Antibiotics: minocydin + rifampin, gentamicin
  • Antimicrobial peptides
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58
Q

Mechanism of Antimicrobial Photochemical Internalization (AM-PCI)

A
  • Antibiotics in endosomes
  • Bacteria in phagosomes

Antibiotics don’t reach bacteria. Releases of antibiotics because of illumination and the response of photosensitizers.

IBIZA – Photo treatment

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59
Q

What is involved in biomaterial-associated infections?

A
  • Biofilms
  • Tissue (intracellular) colonization
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60
Q

What causes the immune dysregulation?

A

Biomaterials together with bacteria.

Can be either pro- or anti-inflammatory.

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61
Q

What is the cause of Lyme Borreliosis (LB)?

A

Caused by Borrelia burgdorferi

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62
Q

How is Borrelia burgdorferi transmitted?

A

By hard bodied Ixodes ticks to humans (via a complex enzootic cycle).

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63
Q

Clinical manifestations of Lyme borreliosis (LB)

A
  • Erythema migrans
  • Lyme carditis
  • Lyme neuroborreliosis (mostly B. garinii)
  • Acrodermatitis chronica atrophicans (chronic skin infection) (mostly B. afzelii)
  • Lyme arthritis (mostly B. burgdorferi)
  • Borrelial lymphocytoma
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64
Q

Most common Borrelia in Europe

A

B. afzelii

Species complexity is higher in Europe B. afzelii, B. aarinii, B. burgdorferi and B bavariensis.

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65
Q

Outer surface protein A

A
  • 31 kDa membrane lipoprotein
  • Expressed by Borrelia in the mid-gut of an unfed tick
  • Tick feeding - OspA down regulation - upregulation of OspC - migration to salivary gland and host
  • Borrelia genospecies are categorized into several serotypes (ST) based on OspA protein i.e. OspA ST1 - ST6
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66
Q

What is the immunogenic part of OspA?

A

C-terminal part is immunogenic and contains protective epitope.

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67
Q

OspA based vaccines are transmission blocking:

A
  1. Tick ingest OspA antibodies while feeding (blood meal) on vaccinated host
  2. Ingested antibodies binds spirochetes expressing OspA in the tick mid-gut before OspA down regulation
  3. Migration to salivary gland is blocked
  4. Spirochete elimination is reported to be independent of host complement*
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68
Q

Mechanism of action of OspA based vaccines

A
  1. rOspA vaccinated human produces IgG anti-OspA antibodies
  2. Tick consumes anti-OspA during feeding on vaccinated human
  3. Anti-OspA antibodies bind to OspA on Borrelia bacteria and kills the bacteria
  4. Migration of the bacteria to tick salivary gland and to the vaccinated human is blocked.
  5. Vaccinated human is protected against LD.
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69
Q

First generation human LB vaccine

A

LYMErix and ImuLyme.

Official reason (fall): lack of demand leading to poor sales.

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70
Q

Second generation OspA vaccines

A
  • Chimeric OspA vaccines
  • OspA ferritin nanoparticle vaccine
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71
Q

VLA15 vaccine OspA (Valneva)

A
  • VLA15 is a combination of 3 proteins
  • Broad protection (pre-clincial murine model) - 4 genospecies representing 6 STs
  • Phase I (first in humans; safety trial) - Phase II (dose optimization trial)
  • Large phase III (efficacy and safety trial, by Pfizer)
  • Attachment of the C-terminals of different OspA ST1 to 6
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72
Q

Initial idea for one protein vaccine against LB came from Italy. What’s the initial idea?

A

Multiple epitopes on one protein as they did for fHBP N. meningitidis (variant 1, 2 and 3).

They took the backbone of variant 1 and added the immunogenic epitopes from variant 2 and 3.

1 protein induced bactericidal antibodies against all meningococcus B strains tested.

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73
Q

Similarity between OspA ST1 - ST6

A

High sequence similarity (identity) was observed between the C-termini of OspA ST1-ST6

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74
Q

Steps in the surface shaping approach - OspA vaccine

A
  • Step 1: sequence conservation analysis ST1-ST6
  • Step 2: homology modeling/structural scaffold. They used B. afzelii ST2 (conserved backbone)
  • Step 3: surface partitioning. Based on monoclonal antibody (mAb) binding to template OspA ST1.
  • Step 4: patch layouts. 6 multivalent vaccines were designed and assessed in animal models.
  • Step 5: serotype distribution.
  • Step 6: mouse model immunogenicity and efficacy analysis.
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75
Q

Immunogenicity and efficacy analysis

A
  1. Immunize C3H/HeN mice with antigen
  2. Collect immune sera and do IgG ELISA, flow cytometry and serum bactericidal assay.
  3. In-vitro grown borrelia or infected tick (ticks off)
  4. Collect final sera and organs and do serology VlsE (variable major protein like sequence expressed) + ELISa
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76
Q

Immunogenicity of the different OspA vaccines

A
  • Variants 1 and 4 (disulfide bond C) – excluded due to low protein yields and lower immune responses
  • Variants 2, 3, 5 and 6 were tested for efficacy in murine models
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77
Q

Final vaccine idea OspA vaccine

A

V3 and V5 linked together with a 23 amino acid long linker sequence P66 protein of B. aarinii strain PBr.

V3-L2-V5 has higher antibody titres than FL-OspA ST1, ST4, ST5 and ST6 and similar to FL-OspA ST2 and ST3.

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78
Q

What is the serum bactericidal titre is defined as?

A

As the reciprocal of the lowest dilution showing 50% bacterial killing.

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79
Q

V3-L2-V5 - single broadly immunogenic and protective OspA vaccine

A
  • High immune response against major OspA STs
  • Highly potent - 100% protection against OspA ST1 and ST2 (significant protection 3 ng dose (ST1))
  • Potential protection against other STs since high immune responses were observed
  • Single antigen vaccine – economically viable (cost-effective)
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80
Q

Tick-born disease

A
  • Ixodes ticks most important vector Northern hemisphere.
  • Carry multiple tick-borne pathogens, among which Borrelia burgdorferi genospecies.
  • Lyme disease incidence increasing.
  • LD diverse clinical manifestations.
  • No human vaccine against LD (anymore/yet)
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81
Q

Pressing issues in the field of tick-borne diseases

A
  • Emergence of other tick-borne diseases (TBEV, BMD, Babesiosis etc.)
  • Need for improved serodiagnostics LD and other TBDs
  • Etiology, diagnosis & treatment of PTLDS (post-treatment LD symptoms)
  • Necessity for a human vaccine to prevent LD.
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82
Q

What immune responses are involved in tick immunity?

A

Cellular immune responses and humoral (IgG) are involved in tick immunity

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83
Q

TSGP1

A
  • 187 AA protein
  • There are homologs in other tick species
  • Top hit protein from Varroa destructor
  • Highly immunogenic (IgG antibodies produced)
  • No effect on tick feeding
  • RNAi of tsgp1 - succesful silencing yet no effect on tick feeding.
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84
Q

Expression of TSGP1 in salivary glands

A

Enhanced TSGP1 expression in salivary glands of ticks infected with Borrelia

85
Q

Syringe inoculation of mice with Borrelia, with or without rTSGP1

A

TSGP1 boosts Borrelia infectivity in mice

86
Q

rTSGP1 vaccination followed by challenge with Borrelia-infected ticks

A

Vaccination against TSGP1 partially protects against Borrelia

87
Q

Human (uninfected) Ixodes tick challenge model (TICK-ME)

A
  • Redness increased after challenges
  • Itch increased
  • Similar weights
  • More dead ticks

T-cell, B-cell, neutrophils, eosinophils present in the tissues.

88
Q

TSGP1 summary

A
  • Induced upon tick feeding
  • Enhanced expression upon Borrelia infection in ticks
  • Boosts Borrelia infectivity in mice
  • Significantly protects against Borrelia when used as an anti-tick vaccine.
89
Q

Plasmodium spp life cycle

A
  • Mosquito
  • Sporozoite
  • In the liver/hepatocyte: schizont
  • Infecting RBCs
  • Asexual blood stage: erythrocyte - ring - trophozoite - schizont - merozoite
  • Gametocyte stage (mosquito drinks this)
  • Microgamete
  • Zygote + macrogamete
  • Ookinete
  • Oocyst
  • Start all over again
90
Q

Mosquitoes that transmit malaria

A

Anopheles

91
Q

Malaria parasites are …

A

Protozoa

Small unicellular parasites

92
Q

Four species of malaria are detected by microscopy

A
  • P. falciparium
  • P. vivax
  • P. ovale
  • P. malariae
93
Q

Five main species of malaria

A
  • P. falciparium
  • P. vivax
  • P. ovale (Curtesi and Walekeri)
  • P. malariae
  • P. knowlesi (zoonosis)
94
Q

Infection with P. vivax, P. ovale or P. malariae

A

Patients can be most ill but infection is not lethal in most cases

95
Q

Infection with P. falciparium

A

Serious disease often with lethal outcome and requires immidiate treatment

96
Q

Difference between P. falciparum and other species

A
  • P. falciparum has high parasitaemia (increasing levels)
  • P. vivax + P. ovale only invade young erythrocytes
  • P. malariae invades old erythrocytes
  • P. falciparum can infect the organs: blocking of small blood vessels i.e. in brain.
97
Q

What molecule mediates cytoadhesion to host cell receptors in malaria?

A

PfEMP1

(Plasmodium falciparum erythrocyte membrane protein 1)

98
Q

Which groups are most at risk of getting malaria ?

A
  1. Children
  2. Non-immune people (travelers)
  3. Pregnant women
99
Q

Evading the immune-system by Plasmodium spp

A

Through antigenic variation

100
Q

The effect of malaria during pregnancy depends on

A
  • Age
  • Gravidity
  • Endemicity
  • HIV status
101
Q

Malaria in pregnancy

A
  • Pregnancy associated malaria: A pregnancy in which malaria infection occurs (can be asymptomatic)
  • Placental malaria: A malaria infection that invests the placenta (and not necessarily the periphery and can be asymptomatic as well (no acute disease)
102
Q

In non-pregnant individuals or in PAM Plasmodium falciparum adheres to … receptor.

A

CD36 receptor

103
Q

Due to antigenic switching plasmodium parasites in pregnancy can bind to … and therefore inhibit a pregnancy specific niche “the placenta” causing placental malaria

A

CSA

104
Q

What is CSA?

A

A sulfated glycosaminoglycan
present on the syncytiotrophoblast in the intervillous space of the placenta, normally functions as a reversible immobilizer for cytokines, hormones and other molecules.

105
Q

In primigravidae

A
  • IgG antibodies of malaria immune primigravidae are not able to recognize the VAR2CSA antigen, because this pregnancy specific antigen differs from malaria antigens expressed in non-pregnant hosts. Primigravidae are therefore more susceptible to placental malaria.
  • Elevated levels of immunosuppressive pregnancy hormone cortisol increase susceptibility in primigravidae
106
Q

What contributes to maternal anemia resulting and subsequently in preterm delivery and post partum hemorrhagic bleeding?

A

Elevated levels of IL-10

107
Q

What might be caused by elevated levels of TNF-a in malaria?

A

Fetal growth restriction might be caused by elevated levels of TNF-a which impair materno–fetal exchanges by damaging placental host tissues.

108
Q

What causes spontaneous abortion by causing fetal necrosis?

A

Elevated levels of TNF-a and IFN-g

Influences uterine contraction and inducing and activating abortion associated NK cells.

109
Q

What causes maternal anemia?

A

Elevated levels of TNF-a

By inflicting oxidative stress on erythrocyte membranes, leading to an increased destruction of these erythrocytes or by its inhibitory effects on erythropoiesis.

110
Q

Accumulation of P. falciparum infected erythrocytes, monocytes and macrophages in the placenta causes …

A

reduction of the intervillous area exposed to maternal blood harming placental blood flow and impairing materno–fetal exchanges (growth restriction).

111
Q

What kind of bacterium is S. aureus?

A

Gram-positive bacterium

112
Q

S. aureus carriage

A
  • Transient versus chronic
  • Nasal carriage ~ 30% of population
  • Presence on skin, pharynx, groin
  • Carriage increases risk of infection
113
Q

S. aureus infections

A
  • Opportunistic pathogen
  • All barrier tissues (skin, lungs etc)
  • Hospital versus community-acquired
  • Surgical-site infections (implants)
114
Q

What ‘immune’ cells are associated with S. aureus infection?

A
  • Neutrophils
  • T-cells (make IL-17 for attracting neutrophils)
115
Q

Neutrophils

A
  • Polymorphic nucleus (3-lobed)
  • ~ 1011 cells/day
  • Granules with hydrolases, peroxidases, defensins, lysozyme and lactoferrin
  • Migration via chemotaxis (IL-8, IL-17, C5a)
116
Q

Two ways in which neutrophils can kill S. aureus

A
  • NETosis (entrapment)
  • Opsonization with antibody or complement binding. Target the bacteria for phagocytosis.
117
Q

Heat-inactivated serum

A

No complement

118
Q

What are the three components that boost neutrophil functions?

A
  1. Antibodies
  2. Complement
  3. T cells
119
Q

Bacterial evasion of neutrophils by S. aureus

A
  1. Evasion of bacterial opsonization
  2. Evasion of phagocytosis and killing
  3. Targeting immune cells
120
Q

Ways in which S. aureus evades bacterial opsonization

A
  • Interfering with antibody binding to bacterial surface (protein A)
  • Inhibition of the complement system (SCIN)
121
Q

Ways in which S. aureus evades phagocytosis and killing

A
  • Neutrophil recruitment to infection site: CHIPS: blocks C5a-receptor
  • Antibody-mediated phagocytosis: FLIPr: blocks Fcy-receptor
  • NET formation and proteases: Eap: blocks elastase and nuclease: degrades NETs
122
Q

Ways in which S. aureus targets immune cells

A

By producing a number of toxins and superantigens.

Toxins
* alpha-toxin: upon binding it oligomerizes and it forms a pore
* Biocomponent pore-forming toxins (leukocidins): F-subunit and S-subunit or LukAB

T cell superantigens
Antigens that can bind outside of the TCR and increases the activation of T cells (~ 25%). Overactivation: hiding in plain sight.

123
Q

Expression of secreted virulence factors: the Agr system

A
  • Accessory gene regulator
  • Quorum sensing
  • Auto-inducing peptides (AIPs)
  • Inhibits adhesion
  • Promotes invasion
124
Q

Hurdles for S. aureus vaccine development

A
  • Immune evasion
  • Translation
  • Strain variation
125
Q

Susceptibility to S. aureus LD50

A

Mice need a lot more S. aureus to get 50% mortality

126
Q

Iron-regulated surface determinant protein B (IsdB) vaccine failed. Why?

A
  • Iron acquisition/metabolism
  • Conserved and immunogenic antigen

Phase 3 clinical trial (ineffective)

  • Prior S. aureus exposure makes the otherwise protective IsdB vaccine non-protective
  • IsdB vaccine recalls non-protective humoral memory from prior *S. aureus * infection
127
Q

Why did the StaphVAX vaccine focusing on the capsular polysaccharide (CPS) failed?

A
  • CPS based (conjugated) vaccines succesful for other bacterial pathogens
  • S. aureus: two dominant capsule (sero-)types CP5 and CP8

Vaccine ineffective in hemodialysis patients.

Only in specific situations and conditions this CPS is present. In vivo no expression.

Dominant MRSA clones do not produce CPS (USA300)

128
Q

Past S. aureus vaccine failures

A
  • Pre-existing immunity
  • Suboptimal vaccine composition
  • Antigenic/phase variation
  • Variation in host susceptibility
129
Q

Considerations for future S. aureus vaccine development

A
  • Immune evasion: neutralization of immune evasion (antibodies)
  • Translation: use pre-clinical models that better resemble human infection
  • Strain variation: target conserved or essential bacterial antigens
130
Q

Theory oral biofilm

A
  1. Formation of a acquired pellicle
  2. Early colonizers that bind to the conditioning layer/pellicle
  3. Late colonizers (all bind F. nucleatum)
  4. Immune system responds to late colonizers

Practice: not that straightforward

131
Q

Brushing can be compared to

A

Ecological succession is the process of change in the species that make up an ecological community over time.

132
Q

What type of cells is colonized with microorganisms in the mouth?

A

Buccal cells

133
Q

Early colonizers thrive in

A

A young biofilm. Oxygen levels are different

134
Q

What are the differences between the early and late biofilms?

A

Lower oxygen levels in late colonizers

135
Q

Conclusion (early colonizers)

A

Human buccal cells and microorganisms associated with them are important in recolonization after brushing
Early colonizers are in fact early proliferators.

136
Q

In a biofilm the geographical distribution of the bacteria is …

A

Not random at all and actually very important.

137
Q

Biofilm is a changing environment. Why?

A
  • Oxygen levels change over time.
  • Anaerobic on the inside of the biofilm

Changing in space and time: spatio-temporal heterogeneity

138
Q

Multi-kingdom biofilms contain

A
  • Fungi
  • Archaea
  • Amoeba
139
Q

Hyphal formation of Candida driver

A

CO2

140
Q

Presence of Candida in the biofilm leads to

A

Oxygen consumption that induces the growth of strict anearobic bacteria.

Switch of metabolism from aerobic to a more anaerobic metabolism (fermentation etc.)

141
Q

Factors that influence the oral microbiome

A
  • Frequent sugar intake leads to selective pressure –> favor aciduric bacteria –> are also acidogenic bacteria
  • Increased caries risk
142
Q

Cariogenic species cycle

A

Sugars –> increase in cariogenic species –> increase in sugar fermentation –> increase in organic acids –> low pH –> higher caries risk and selection for cariogenic species –> cycle….

143
Q

What do all bacteria fight for?

A

Iron

Heme group is causing the red fluorescence

144
Q

Cycle of periodontitis and gingivitis

A

Accumulation of bacteria –> increased inflammatory mediators –> gingival inflammation –> increase in GCF leakage (gingival crevicular fluid) –> increase in protein and increase in iron –> favorable conditions for proteolytic bacteria + keystone pathogen mechanisms –> more inflammatory mediators –> cycle…

145
Q

Oral diseases … Koch’s postulates

A

Defy

146
Q

Definition of the microbiome

A

Total of all micro-organisms in a specific location.
Micro-organisms: bacteria, fungi, protozoa and viruses.

Microbiota + the theatre of activity (microbial structural elements and microbial metabolites)

147
Q

What’s the oralome?

A

Host and oral microbiome combined (in balance; homeostasis)

Healthy oralome (eubiosis) or an unbalanced oralome (dysbiosis)

148
Q

Diseases associated with an unbalanced oralome (dysbiosis)

A
  • Periodontitis
  • Endocarditis
  • Atherosclerosis
  • Alzheimer’s Disease
  • Diabetes
  • Head and Neck Cancer
149
Q

Prediction of disease?

A

Detection of a dysbiosis at an early time point

150
Q

How to define a healthy oral cavity?

A

No caries, no gingivitis, no erosion, no periodontitis, no mucositis, no halitosis.

151
Q

Origin of colonization (oral microbiome)

A

The mother. Colonization starts at birth. Transmission through breast-feeding.

152
Q

Pregnancy gingivitis

A

Hormonally driven gingivitis (not caused by bacteria).

Bacteria will translocate to the placenta. Tregs recognize mother’s microbiome as ‘safe’ are generated in fetal lymphoid tissue.

153
Q

Hierarchy of regulatory mechanisms

A
  1. DNA structure: presence, amplification, rearrangement, modification (porA/pili)
  2. Transcriptional regulation: activation, repression (Sigma factors, Fur)
  3. Post-transcriptional regulation: translation repression, transcript stability (sRNA, riboswitch, RNAT)
154
Q

What is the definition of an operon?

A

Gene that has a common promoter.

A set of neighboring genes in a genome that is transcribed as a single polycistronic message.

155
Q

What is the definition of an regulon?

A

A set of target genes regulated by the same transcription factor by physically binding to regulatory motifs to accomplish a specific biological function.

They use the same common regulator.

156
Q

What is the definition of a stimulon?

A

All the genes whose expression is increased or decreased by a specific external stimulus.

157
Q

What are the reasons for fine tuning expression of genes?

A
  1. Essential to adapt to growth conditions encountered in the host.
  2. To express bacterial virulence factors that are essential components for pathogenesis and enable the host to survive.
158
Q

DNA/Gene order of ‘parts’

A
  • Promoter (-35 . spacer (17 nt) . -10)
  • Start site transcription
  • A/T rich enhancer
  • Shine Dalgarno (SD) - ribosomal binding site (RBS)
  • Start site translation
  • Protein encoding gene (A/C rich codons)

5-UTR consists of everything between start site transcription and the start site translation.

159
Q

What is slipped strand mispairing?

A

Illegitimate base pairing in regions of repetitive DNA during replication: production of deletions or insertions of repeat units.

Example: porA gene of N. meningitidis has expression variants of the outer membrane protein

160
Q

What determines the binding-strength of RNA polymerase?

A

The length of the DNA stretch (spacer) between the -35 and -10 box.

161
Q

Essential for all organisms to grow

A

Iron

162
Q

What are sigma factors?

A
  1. In direct contact with the DNA
  2. Recognize specific promoter consensus sequences (-35 and -10 region)

Is complexed with RNA polymerase:
* Sigma 70 - general (housekeeping)
* Sigma 32 - stress
* Sigma E - heat shock
* Sigma 54 - nitrogen

A sigma factor (σ factor or specificity factor) is a protein needed for initiation of transcription in bacteria.

163
Q

Transcriptional regulators in general:

A
  • Recognize specific sequences in or near promoter region
  • Have different modes of action

Repressor proteins: steric hindrance, blocking elongation, DNA looping

Activator proteins: class I activation, class II activation, activation by conformation change

164
Q

Transcriptional regulation by sigma factors

A
  • Specific promoter sequence recognition: sigma factors
  • Specific transcriptional regulators: Fur (Ferric Uptake Regulator)
  • Specific stimuli: Fe

50% of iron responsive genes are under control of Fur

165
Q

What is Fur and how does it work?

A

Fur is a dimeric protein that has a high affinity for iron. Genes regulated by Fur are characterized by: consensus sequence: Fur box or Fbs (Fur binding site) (partially overlapping with promoter sequence)

166
Q

What is the Fur box DNA consensus of meningococcus

A

NATWATNATWATNATWAT

167
Q

Explain the two conditions for Fur

A
  • Excess iron: there’s no need for the uptake of iron-complexed proteins. Fur binds Fe and is active. Active Fur binds to the Fur-box (no transcription)
  • Low iron: bacteria need iron, so expression of genes coding for receptors binding iron-complexed proteins on! Fur can’t bind Fe which leads to a conformation change (in-active Fur). RNA polymerase can bind the Fur-box and there transcription of the coding region.

Fur related genes are crucial for iron acquisition.

168
Q

Example of Fur related genes

A
  • Lactoferrin
  • Transferrin
  • Binding proteins
169
Q

What are small RNAs (sRNAs)

A
  • sRNA (20-500 nt) encoded in the intergenic region or on anti-sense strand
  • Regulate stability and translation of mRNAs, often in conjunction with the conserved prokaryotic RNA chaperone protein Hfq
  • Antisense to the untranslated 5’ region (5-UTR) of the target mRNA to be regulated.
170
Q

How is the expression of sRNAs often induced?

A

By environmental stress.

Target mRNAs often encode for stress responsive components. One sRNA can regulate more mRNA targets

171
Q

What do some sRNAs contain?

A

A Fur box in their promoter region and ar Fur/iron regulated.

172
Q

Four mechanisms of riboregulation

A
  1. Translation repression by sRNAs
  2. Translation activation by sRNAs
  3. mRNA degradation because of the binding of sRNAs
  4. Stabilizing mRNA because sRNA is bound (no ribonuclease able to bind anymore)
173
Q

What are riboswitches?

A

Riboswitches are located in the 5-UTR of the mRNAs. They change the structure of RNA in response to binding of a specific metabolite:
* Guanidine
* Adenosine
* Lysine
* SAM
* Mg

mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding.

174
Q

The different parts of riboswitches

A
  • Aptamer: performs ligand recognition
  • Expression platform: regulates either transcription or translation initiation

Some riboswitches also control mRNA decay

175
Q

Ligand binding to riboswitches either

A

Sequesters (OFF switch)

or

Frees (ON switch) the RBS

176
Q

What are RNA thermometers (RNAT)?

A

RNATs are elements usually located in the 5-UTR of the mRNAs. They operate by changing structure in response to temperature fluctuation by a zipper like mechanism.

Regulate expression of virulence genes of pathogens.

177
Q

Give an example of a RNAT

A

prfA Listeria that works together with riboswitch and sRNA. sRNA is transcribed from a riboswitch and controlled by a RNAT

178
Q

Almost all antibiotics are based on …

A

Antimicrobial compounds produced by micro-organisms, especially bacteria.

179
Q

Three reasons for AMR problem

A
  1. Uncontrolled (over)use
  2. Resistance spread by horizontal gene transfer (natural genetic competence, phage transduction and conjugation)
  3. Lack of development of novel classes antibiotics
180
Q

Reasons for lack of antibiotic development

A

Too expensive since production is cheap, use is limited in time. Yet the development is as costly as any internal medicine (clinical trials phase I - III 100 - 500 106 euro)

181
Q

What are mode of action (MOA) studies?

A

The study of its activity

181
Q

One of the tools for MOA is …

A

Cytological profiling

182
Q

Daptomycin is a lipopeptide that targets bacterial membranes. What are the two proposed functions?

A

Membrane depolarization or blocking of the cell wall synthesis.

183
Q

What did bacterial cytological profiling of daptomycin show?

A
  • Bacterial cytological profiling showed delocalization of certain peripheral membrane proteins
  • No strong membrane depolarization
  • Daptomycin clusters fluid lipids (lipids with short and/or unsaturation and/or branched fatty acids)
  • This leads to detachment of peripheral membrane proteins (e.g. those with amphipathic alpha helix membrane anchors). Including essential N-acetylglucosamine transferase MurG involved in peptidoglycan synthesis precursor synthesis lipid II.
184
Q

What did bacterial cytological profiling of tetracycline show?

A

Tetracycline binds to ribosome blocking translation.

  • TEM (transmission electron microscopy) shows that tetracycline disturbs the cell membrane
  • Bacterial cell biology shows that tetracycline sits mostly in the cell membrane
  • Tetracycline can disturb the localization of certain peripheral membrane proteins, thus has an additional MOA
185
Q

What are persister cells?

A
  • Persister cells are antibiotic tolerant (not the same as antibiotic resistant!)
  • Cause of recurrent infections and resistance
  • Tolerant because they do not grow, or grow very slowly
  • Non growing cells occur in every bacterial culture
  • Persisters are an example of cellular heterogeneity in bacterial populations, which is common in bacterial cultures
186
Q

What is the reason that antibiotics work despite the presence of persister cells?

A

That hey are removed by the immune system.

187
Q

Give an example of regulated cellular heterogeneity in bacterial cultures

A

Bistable regulation (also called bimodal regulation.

Other example: phenotypic switching due to phase variation, e.g. formation of different surface proteins and/or carbohydrates to evade the immune system)

188
Q

Natural genetic competence in Bacillus subtilis

A
  • Regulation mechanism is simple and only requires (i) an autostimulatory loop
  • Threshold level for the activity of this loop (often cooperative binding of a transcription factor to DNA)
  • Stochastic fluctuations in gene expression (also called noise in gene expression)
189
Q

What is the function of bistable regulation?

A
  • Bet-hedging
  • Division of labour
190
Q

Mechanisms that lead to a subpopulation of persisters are

A
  • Nutrient reduction in biofilms
  • Stringent response
  • SOS response
  • Toxin-antitoxin systems
191
Q

What is a novel antibiotic target to kill persisters?

A

Persister cells still have a membrane potential.

192
Q

B. subtilis persisters

A
  • Dissipation of membrane potential (by valinomycin) causes ROS (reactive oxygen species) that contribute to the killing including causing DNA damage
  • Many antibiotics cause ROS, primarily hydroxyl radicals likely due to release of Fe2+ ions from iron-sulfur containing proteins, triggering the Fenton reaction
    Fenton reaction: hydrogen peroxide conversion catalyzed by Fe2+ ions into hydroxyl radicals
  • However, not the case with depolarization using valinomycin, since iron chelators do not mitigate the effect
    moreover, superoxide dismutase mutant are sensitive, but katalase mutant not, indicating that superoxide radicals are formed and not hydroxyl radicals
  • Confirmed by using specific superoxide radical and hydroxyl radical scavengers
  • Electron transfer chain (ETC) well know source of ROS (also in mitochondria)
  • Only ETC mutant that mitigated the effect of valinomycin was inactivation of the conserved Rieske protein (QcrA), which is also present in mitochondria
  • Rieske protein contains Fe2+ and is a well know source of superoxide radicals in mitochondria
  • Bacterial cell biology experiments showed that this protein detaches from the other components of the Cytochrome BC complex (QcrB, QcrC)

In TB inactivation of Rieske protein increases the resistance to membrane dissipating antibiotics (now explained).

Many bacteria do not contain Rieske protein but their persisters are still killed by membrane depolarization, likely linked to ROS formation. How is not know (but important to discover)

193
Q

Predominant pathogens in biomaterial-associated infection (BAI)

A

Staphylococcus epidermidis (CoNS) and S. aureus

194
Q

What is the function of suturing?

A

With sutures way less S. aureus infection than in people without sutures.

195
Q

Biofilm formation: 5 steps. What are the three most important ones?

A
  1. Adherence
  2. Maturation/growth
  3. Detachment
196
Q

Characteristics of biofilms

A
  • Quorum sensing
  • Difficult to phagocytose
  • Not effectively reached by all antibiotics
  • In dormant state, less susceptible to antibiotics
  • Persisters
  • A persisting inflammatory stimulus
197
Q

Sepvp catheter

A

Too strong pro-inflammatory response around Sepvp catheter.

198
Q

Pvp-coated polyamide catheter

A

Too little immune activation around Pvp-coated polyamide catheter.

  • Hydrophilic surface
  • In vitro 75% less bacterial adherence than on PA
199
Q

Pathogenesis of implant-associated sepsis

A

Disbalance of pro-/anti-inflammatory cytokines: low IFN-y

Macrophage survival and multiplication leading to:
1. Spreading
2. Systemic disease
3. Sepsis

200
Q

Goal for biomaterial

A

Develope materials which even in presence of micro-organisms:
* will allow a proper host immune response capable of clearing the pathogens
* will allow proper host tissue responses resulting in infection-free wound healing

201
Q

Material characteristics (preferably)

A
  • Chemistry of base materials: bio-compatibility, “immune-compatibility”
  • Surface topographies: host response depending on interaction with surface
  • Coatings to optimize host response
202
Q

Release systems (preferably) for biomaterials

A

Release of antimicrobials
* Steering the host response: release of inflammatory / anti-inflammatory mediators
* SMART release: the right actives at the right time, response to environmental triggers

203
Q

Biomaterial-associated infection (BAI)

A
  • Mostly caused by Staphylococci
  • Biofilm formation on the implant
  • Colonization of the peri-implant tissue
204
Q

In the presence of titanium S. epidermidis infection

A

persists

205
Q

What do the temporal gene expression patterns show?

A

Distinct differences:
* Early response due to infection
* Later and longer response due to material
* Combination leads to strong early response –> immune-dysregulation?

206
Q

Domains point to function

A
  • Anti-apoptosis
  • Ubiquitylation
  • Lipid metabolism
  • Membrane trafficking
207
Q
A

Advanced Medical Microbiology (2 decks)

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