Professor Zhang Tiehua of Jilin University, et al.: Research progress on the effect of toxin-antitoxin system on the formation of non-cultureable microbial activity

The survival of microorganisms is challenged by pressure from the host as well as the natural environment, especially by the changing natural environment (such as low temperature, high temperature, heavy metals, high osmotic pressure, disinfectants, antibiotics, etc
.
).
In order to adapt to harsh environments, some microorganisms choose to form a life form with low metabolic activity but high resistance, such as spores, live non-culturable (VBNC) status, retained bacteria, etc
.
Sporozoa is a dormant body
formed in the bacterium by spor-producing bacteria (mainly Bacillus and Clostridium species) when environmental nutritional conditions are lacking.
The spores have extremely low water content and strong stress resistance, and the food industry also measures the sterilization effect of the product by the killing effect
of the spores.
VBNC state and retention state are a dormant state
formed by non-sporulating bacteria in the face of adverse environments.
Retained bacteria are tolerant to multiple drugs, leading to treatment failure
.
Pathogenic and spoilage bacteria in VBNC state still have the risk of spoilage and disease, and when conditions are right, they may return to a cultureable state, which poses a serious threat
to the food industry and human health.

The toxin-antitoxin (TA) system consists of two co-expressed genes, one encoding a toxin and the other encoding an antitoxin
.
Toxins can inhibit bacterial growth, while antitoxins neutralize toxins and inhibit their toxic effects
.
In harsh environments, the TA system is activated and involved in regulating bacterial growth
.
Studies have shown that the TA system is involved in the formation
of microbial retention state and VBNC state.
However, the activation mode of TA system in adverse environment and the relationship between TA system and VBNC are not comprehensive
.
Tiehua Zhang, Lingling Meng and Fengfeng Zhao* from the College of Food Science and Engineering, Jilin University, will focus on the relationship between
TA system and microbial VBNC state formation.

1.
VBNC state and retention state of microorganisms

The VBNC state is a dormant state formed when non-sporulating bacteria are in a bad environment, and is a survival strategy adopted by microorganisms (Figure 1).

At present, more than 100 microorganisms have been found to be able to form VBNC status, including a variety of foodborne pathogenic bacteria and spoilage bacteria
.
Traditional bacterial culture methods are considered the gold standard for the detection of foodborne pathogens, but the non-culturability of VBNC-status bacteria makes this culture-based assay undetectable VBNC-state bacteria, which leads to a greatly underestimation of bacterial content in food, resulting in microbial risk
.
At present, the determination of VBNC cell number is based on indirect methods, that is, obtained indirectly by measuring the number of viable cells of microorganisms and the number of cells that can be cultured, and the difference between the two is the number
of VBNC cells.

At present, the factors that can induce microorganisms to enter the VBNC state include temperature, nutrition, pH value, osmotic pressure, oxygen concentration, heavy metals, antibiotics, disinfectants, detergents, preservatives, etc.
; In addition, food processing processes such as drying, pasteurization, ultraviolet irradiation, high-pressure carbon dioxide, pulsed electric field, ultrasonic, and thermal ultrasound can also induce microorganisms into the VBNC state
.
The more common characteristics of VBNC state microorganisms include cell morphological changes, decreased metabolic activity, increased resistance to external stimuli, and changes in gene expression
.
When conditions are suitable, such as reversing induction stress, adding chemicals, co-culturing with host cells, or adding the resuscitation promoter Rpf, some VBNC-state microorganisms are able to revive and regain the ability to
grow and reproduce on the medium.

Antibiotic retention bacteria are a subset of cells
produced during antibiotic treatment that are resistant to antibiotics.
Unlike resistant cells, which carry heritable resistance factors, antibiotic retention bacteria typically exhibit low metabolic activity during antibiotic treatment, which allows them to tolerate antibiotics
without the need to undergo genetic mutations.
In 2015, Ayrapetyan et al.
proposed the dormant continuum hypothesis (Figure 2), which suggests that the retention state of microorganisms is closely related
to the VBNC state.
The hypothesis states that certain environmental stresses, such as antibiotic treatment, induce the degradation of antitoxins in cells, resulting in the release of their homologous toxins (red triangle in Figure 2), which can affect cell growth
by affecting cell metabolism.
In the early stage of environmental stress treatment, tolerant non-growth cells - retainer bacteria are produced, which can quickly resume growth
after the environmental stress is eliminated.
However, if the cells are exposed to environmental stress for a long time, the amount of free toxins accumulated will increase, further reducing the metabolic activity of the cells, resulting in increased dormancy, and the cells entering the VBNC state
.
When environmental stress is removed, cells in the VBNC state take longer to recover
than retained bacteria.

In the process of studying the formation mechanism of VBNC state of Staphylococcus aureus, a protein was isolated from VBNC cell inducer solution, and it was found that the protein belonged to a toxin protein through sequence alignment, and it was experimentally proved that this protein significantly affected the formation
of VBNC cells.
At present, a large number of studies have proved that the TA system is involved in the formation of retaining bacteria, but the relationship between the TA system and the formation of VBNC state is still rare
.
In this paper, the effect
of TA system on the formation of microbial VBNC state will be reviewed from the expression of TA trigger system and TA system itself in VBNC state microorganisms and their effect on VBNC state formation.

2.
TA system overview

According to the chemical composition of toxins and antitoxins and the way they interact, TA systems can be divided into 8 types, including 6 widely reported TA systems and 2 newly proposed TA systems, which are named I~VIII TA systems
.
Among them, the antitoxin of type I, III and type VIII TA systems is RNA, and the antitoxin in the remaining five TA systems are proteins; In addition, except for the newly discovered type VIII TA system toxins are RNA, the toxins of the rest of the TA system are proteins
.
Among these 8 TA systems, antitoxin inhibits toxins in various ways, which can be summarized as inhibiting the translation of toxin proteins, directly binding to toxins and affecting their function, interfering with the binding of toxins to targets, acting as adapters to promote the degradation of toxin proteins, and modifying toxins
.
The mode of action of antitoxins and toxins in each TA system is introduced
.
In the type I TA system, the antitoxin is RNA, and its sequence is complementary to the mRNA sequence encoding the toxin protein, so the antitoxin can bind to the mRNA encoding the toxin protein, inhibit toxin translation, and thus play a role
in regulating toxin expression.
In type II TA systems, toxins and antitoxins are both proteins, and antitoxin proteins inhibit the toxicity of toxins by binding to toxin proteins with affinity
.
In type III TA systems, antitoxins are RNA that act directly on toxin proteins to inhibit toxin activity
.
In the type IV TA system, antitoxin is a protein, but it cannot directly bind to toxin proteins, but indirectly inhibits the function
of toxin proteins by interacting with the target of toxin proteins.
In the V-type TA system, antitoxin is a ribonuclease (RNase) that specifically cleaves mRNA encoding toxin proteins, preventing its translation
.
In the Type VI TA system, the antitoxin SocA acts as an adapter to promote the degradation
of the toxin protein SocB by casein lyase (Clp) XP.
Different from the above 6 types, in the three TA systems of Hha/TomB, TglT/TakA and HepT/MntA, antitoxin (TomB, TakA, MntA) exerts antagonism to toxin proteins by modifying its homologous toxins (Hha, TglT, HepT), and the researchers suggest that this type of TA system be named "Type VII TA System"
。 In the Hha/TomB TA system of Escherichia coli, the antitoxin TomB can oxidize the cysteine residues conserved by the toxin protein Hha, thereby inactivating the toxin protein; In the TglT/TakA TA system of Mycobacterium tuberculosis, the antitoxin TakA is a novel atypical serine protein kinase that inhibits the activity of toxin proteins by phosphorylating the S78 site of the toxin protein TglT.
In the HepT/MntA TA system, which is widely present in bacteria and archaea, MntA antitoxin has nucleic transferase activity, which can transfer 3 AMPs to tyrosine residues near the active site of the toxin protein HepT, inhibiting the role of
toxins.
In type VIII TA systems, the toxin SdsR (also known as RyeB) and the antitoxin RyeA are sRNAs encoded by positive and negative strands of DNA at the same site, so the antitoxin can complement the toxin to form base pairs to neutralize the role of
the toxin.
But there are not only these 8 members of
the TA system.

3.
The influence of TA system on the formation of VBNC state

TA triggers the effect of the system on VBNC state formation

The TA trigger system includes 8 genes
: spoT, relA, relE, clpA, clpP, lon, ppk and ppx.
Studies have shown that relA, spoT, lon, ppx, and ppk in TA-triggered systems are related
to the formation of microbial VBNC states 。 relA gene encodes the synthase protein RelA of the signaling molecule guanosine tetraphosphate (ppGpp), ppGpp can sense environmental stress, regulate DNA replication and transcription, and is a stress factor that can induce microorganisms to enter the retention state and VBNC state.
The SpoT protein encoded by the spoT gene has ppGpp hydrolase activity and weak synthase activity.
Lon encoded Lon protease is an ATP-dependent protease that can degrade type II antitoxin; ppx encodes exocytophosphatase (PPX), the classical PPX is monofunctional, which can maintain polyphosphate (PolyP) homeostasis, and the other is bifunctional PPX/guanosine pentaphosphate hydrolase (PPX/GPPA), PPX/GPPA enzyme has both PPX and GPPA activities, responsible for the production of ppGpp; ppk encodes polyphosphate kinase (PPK), which mediates the synthesis of PolyP, which activates Lon protease to degrade antitoxins
。 Zhang Jingfeng et al.
used ampicillin to induce Cronobacter sakazakii into the VBNC state, and compared the transcription levels of six genes, Enterobacter sakazakii rpoS, spoT, relA, ppx, ppk, and lon in the logarithmic growth phase and VBNC state, and found that the expression of relA, ppx, ppk, lon, and rpoS genes in VBNC cells was significantly upregulated.
However, the expression of spoT gene in VBNC cells did not change significantly, so the possible mechanism of VBNC state formation of Enterobacter sakazakii was proposed, that is, RelA and PPX/GPPA acted to generate ppGpp, ppGpp accumulates PolyP by activating PPK, and the accumulated PolyP can further activate Lon protease to degrade antitoxin, and the free toxin released therefrom induces Enterobacter sakazakii to enter the VBNC state
.

Effects of toxins and antitoxins in the TA system on the formation of VBNC states

This article summarizes the possible influence mechanism of TA system on the formation of microbial VBNC state (Figure 3): in an adverse environment, TA triggers the differential expression of genes in the system, causing the degradation of antitoxin, resulting in the imbalance of toxin-antitoxin ratio and increasing the content of free toxins, which promote the formation
of microbial VBNC state by inhibiting DNA replication and protein translation, promoting mRNA break, and inhibiting cell division.

Conclusion